Dgat genes from oleaginous organisms for increased seed storage lipid production and altered fatty acid profiles in oilseed plants

ABSTRACT

Transgenic soybean seed having increased total fatty acid content of at least 10% and altered fatty acid profiles when compared to the total fatty acid content of non-transgenic, null segregant soybean seed are described. DGAT genes from oleaginous organisms are used to achieve the increase in seed storage lipids.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.13/328,677, filed Dec. 16, 2011, which is a divisional of U.S.application Ser. No. 12/470,517, now U.S. Pat. No. 8,153,859, filed May22, 2009, which claims the benefit of U.S. Provisional Application No.61/055,585, filed May 23, 2008, the contents of which are herebyincorporated by reference.

FIELD OF THE INVENTION

This invention is in the field of biotechnology, in particular, thispertains to polynucleotide sequences encoding diacylglycerolacyltransferase genes and the use of these acyltransferases forincreased seed storage lipid production and altered fatty acid profilesin oilseed plants.

REFERENCE TO A SEQUENCE LISTING SUBMITTED AS A TEXT FILE VIA EFS-WEB

The official copy of the sequence listing is submitted electronicallyvia EFS-Web as an ASCII formatted sequence listing with a file named430124SEQLIST.txt, created on Feb. 15, 2013, and having a size of 1436kilobytes and is filed concurrently with the specification. The sequencelisting contained in this ASCII formatted document is part of thespecification and is herein incorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

Plant lipids have a variety of industrial and nutritional uses and arecentral to plant membrane function and climatic adaptation. These lipidsrepresent a vast array of chemical structures, and these structuresdetermine the physiological and industrial properties of the lipid. Manyof these structures result either directly or indirectly from metabolicprocesses that alter the degree of unsaturation of the lipid. Differentmetabolic regimes in different plants produce these altered lipids, andeither domestication of exotic plant species or modification ofagronomically adapted species is usually required to produceeconomically large amounts of the desired lipid.

There are serious limitations to using mutagenesis to alter fatty acidcomposition and content. Screens will rarely uncover mutations that a)result in a dominant (“gain-of-function”) phenotype, b) are in genesthat are essential for plant growth, and c) are in an enzyme that is notrate-limiting and that is encoded by more than one gene. In cases wheredesired phenotypes are available in mutant crop lines, theirintrogression into elite lines by traditional breeding techniques isslow and expensive, since the desired oil compositions are likely theresult of several recessive genes.

Recent molecular and cellular biology techniques offer the potential forovercoming some of the limitations of the mutagenesis approach,including the need for extensive breeding. Some of the particularlyuseful technologies are seed-specific expression of foreign genes intransgenic plants [see Goldberg et al (1989) Cell 56:149-160], and theuse of antisense RNA to inhibit plant target genes in a dominant andtissue-specific manner [see van der Krol et al (1988) Gene 72:45-50].Other advances include the transfer of foreign genes into elitecommercial varieties of commercial oilcrops, such as soybean [Chee et al(1989) Plant Physiol. 91:1212-1218; Christou et al (1989) Proc. Natl.Acad. Sci. U.S.A. 86:7500-7504; Hinchee et al (1988) Bio/Technology6:915-922; EPO publication 0 301 749 A2], rapeseed [De Block et al(1989) Plant Physiol. 91:694-701], and sunflower [Everett et al (1987)Bio/Technology 5:1201-1204], and the use of genes as restrictionfragment length polymorphism (RFLP) markers in a breeding program, whichmakes introgression of recessive traits into elite lines rapid and lessexpensive [Tanksley et al (1989) Bio/Technology 7:257-264]. However,application of each of these technologies requires identification andisolation of commercially-important genes.

Most free fatty acids become esterified to coenzyme A (CoA), to yieldacyl-CoAs. These molecules are then substrates for glycerolipidsynthesis in the endoplasmic reticulum of the cell, where phosphatidicacid and diacylglycerol (DAG) are produced. Either of these metabolicintermediates may be directed to membrane phospholipids (e.g.,phosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine) orDAG may be directed to form triacylglycerols (TAGs), the primary storagereserve of lipids in eukaryotic cells.

Diacylglycerol acyltransferase (“DGAT”) is an integral membrane proteinthat catalyzes the final enzymatic step in the production oftriacylglycerols in plants, fungi and mammals. This enzyme isresponsible for transferring an acyl group from acyl-coenzyme-A to thesn-3 position of 1,2-diacylglycerol (“DAG”) to form triacylglycerol(“TAG”). DGAT is associated with membrane and lipid body fractions inplants and fungi, particularly, in oilseeds where it contributes to thestorage of carbon used as energy reserves. TAG is believed to be animportant chemical for storage of energy in cells. DGAT is known toregulate TAG structure an direct TAG synthesis. Furthermore, it is knownthat the DGAT reaction is specific for oil synthesis.

TAG is the primary component of vegetable oil in plants, It is used bythe seed as a stored form of energy to be used during seed germination.

Two different families of DGAT proteins have been identified. The firstfamily of DGAT proteins (“DGAT1”) is related to the acyl-coenzymeA:cholesterol acyltransferase (“ACAT”) and has been described in U.S.Pat. Nos. 6,100,077 and 6,344,548. A second family of DGAT proteins(“DGAT2”) is unrelated to the DGAT1 family and is described in PCTPatent Publication WO 2004/011671 published Feb. 5, 2004. Otherreferences to DGAT genes and their use in plants include PCT PublicationNos. WO2004/011,671, WO1998/055,631, and WO2000/001,713, and US PatentPublication No. 20030115632.

Applicants' Assignee's copending published patent application US2006-0094088 describes genes for DGATs of plants and fungi and their useis in modifying levels of polyunsaturated fatty acids (“PUFAs”) inedible oils.

Applicants' Assignee's published PCT application WO 2005/003322describes the cloning of phosphatidylcholine diacylglycerolacyltransferase and DGAT2 for altering PUFA and oil content inoleaginous yeast.

SUMMARY OF THE INVENTION

The present invention concerns a transgenic soybean seed havingincreased total fatty acid content of at least 10% when compared to thetotal fatty acid content of a non-transgenic, null segregant soybeanseed.

In a second embodiment, the present invention concerns a method forincreasing the total fatty acid content of a soybean seed comprising:

(a) transforming at least one soybean cell with a recombinant constructhaving at least one DGAT sequence;

(b) selecting the transformed soybean cell(s) of step (a) having anincreased total fatty acid content of at least 10% when compared to thetotal fatty acid content of a non-transgenic, null segregant soybeanseed.

In a third embodiment, the present invention concerns a transgenic cornkernel having increased total fatty acid content of at least 10% whencompared to the total fatty acid content of a non-transgenic, nullsegregant corn kernel.

In a fourth embodiment, the present invention concerns a method forincreasing the total fatty acid content of a corn kernel comprising:

(a) transforming at least one corn kernel with a recombinant constructhaving at least one DGAT sequence;

(b) selecting the transformed corn kernel(s) of step (a) having anincreased total fatty acid content of at least 10% when compared to thetotal fatty acid content of a non-transgenic, null segregant cornkernel.

In a fifth embodiment, the present invention concerns a transgenicsoybean seed having increased total fatty acid content of at least 10%and an increased oleic acid content of at least 25% when compared to thetotal fatty acid content and oleic acid content of a non-transgenic,null segregant soybean seed.

In a further embodiment, the present invention concerns a transgenicsoybean having increased total fatty acid content of at least 10% and atleast any one of i) an increased oleic acid content of at least 25%; ii)a decreased linolenic acid content of at least 25%; iii) a decreasedlinoleic acid content of at least 4%; iv) a decreased palmitic acidcontent of at least 8%; and v) an increased stearic acid content of atlease 14% when compared to the total fatty acid content and oleic,linolenic acid, linoelic acid, palmitic acid or stearic acid,respectively, content of a non-transgenic, null segregant soybean seed.

In an sixth embodiment, the present invention concerns a method forincreasing the total fatty acid content and oleic acid content of asoybean seed comprising:

(a) transforming at least one soybean cell with a recombinant constructhaving at least one DGAT sequence;

(b) selecting the transformed soybean cell(s) of step (a) having anincreased total fatty acid content of at least 10% and an increasedoleic acid content of at least 25% when compared to the total fatty acidcontent and oleic acid content of a non-transgenic, null segregantsoybean seed.

In a seventh embodiment, the present invention concerns a method forincreasing the total fatty acid content and decreasing linolenic acidcontent of a soybean seed comprising:

(a) transforming at least one soybean cell with a recombinant constructhaving at least one DGAT sequence;

(b) selecting the transformed soybean cell(s) of step (a) having anincreased total fatty acid content of at least 10% and a decreasedlinolenic acid content of at least 25% when compared to the total fattyacid content and oleic acid content of a non-transgenic, null segregantsoybean seed.

In an eighth embodiment, the present invention concerns a method forincreasing the total fatty acid content and decreasing linoleic acidcontent of a soybean seed comprising:

(a) transforming at least one soybean cell with a recombinant constructhaving at least one DGAT sequence;

(b) selecting the transformed soybean cell(s) of step (a) having anincreased total fatty acid content of at least 10% and a decreasedlinoleic acid content of at least 4% when compared to the total fattyacid content and oleic acid content of a non-transgenic, null segregantsoybean seed.

In a ninth embodiment, the present invention concerns a method forincreasing the total fatty acid content and decreased palmitic acidcontent of a soybean seed comprising:

(a) transforming at least one soybean cell with a recombinant constructhaving at least one DGAT sequence;

(b) selecting the transformed soybean cell(s) of step (a) having anincreased total fatty acid content of at least 10% and a decreasedpalmitic acid content of at least 8% when compared to the total fattyacid content and oleic acid content of a non-transgenic, null segregantsoybean seed.

In a tenth embodiment, the present invention concerns a method forincreasing the total fatty acid content and stearic acid content of asoybean seed comprising:

(a) transforming at least one soybean cell with a recombinant constructhaving at least one DGAT sequence;

(b) selecting the transformed soybean cell(s) of step (a) having anincreased total fatty acid content of at least 10% and an increasedstearic acid content of at least 14% when compared to the total fattyacid content and oleic acid content of a non-transgenic, null segregantsoybean seed.

Any of the transgenic seed of the invention may comprise a recombinantconstruct having at least one DGAT sequence which can be selected fromthe group consisting of DGAT1, DGAT2 and DGAT1 in combination withDGAT2. Furthermore, the DGAT sequence can be an oleaginous organismsequence.

Also within the scope of the invention are product(s) and/orby-product(s) obtained from the transgenic soybean seeds of theinvention.

In a tenth embodiment the present invention concerns an isolatedpolynucleotide comprising:

(a) a nucleotide sequence encoding a polypeptide having diacylglycerolacyltransferase activity wherein the polypeptide has at least 80% aminoacid identity, based on the Clustal V method of alignment, when comparedto an amino acid sequence as set forth in SEQ ID NOs: 135, 136, 147,162, 176, 215, 234, 265, 272, 299, 304, 306, 308, 310, 312, 314, 316,318, 320, 322, 351, or 363;

(b) a nucleotide sequence encoding a polypeptide having diacylglycerolacyltransferase activity, wherein the nucleotide sequence has at least80% sequence identity, based on the BLASTN method of alignment, whencompared to a nucleotide sequence as set forth in SEQ ID NO: 133, 134,146, 161, 175, 214, 233, 264, 271, 298, 303, 305, 307, 309, 311, 313,315, 317, 319, 321, 350, or 362:

(c) a nucleotide sequence encoding a polypeptide having diacylglycerolacyltransferase activity, wherein the nucleotide sequence hybridizesunder stringent conditions to a nucleotide sequence as set forth in SEQID NO: 133, 134, 146, 161, 175, 214, 233, 264, 271, 298, 303, 305, 307,309, 311, 313, 315, 317, 319, 321, 350, or 362; or

(d) a complement of the nucleotide sequence of (a), (b) or (c), whereinthe complement and the nucleotide sequence consist of the same number ofnucleotides and are 100% complementary.

It can also be appreciated that the present invention concerns anisolated polynucleotide comprising:

(a) a nucleotide sequence encoding a polypeptide having diacylglycerolacyltransferase activity wherein the polypeptide is set forth in SEQ IDNOs:135, 136, 147, 162, 176, 215, 234, 265, 272, 299, 304, 306, 308,310, 312, 314, 316, 318, 320, 322, 351, or 363;

(b) a nucleotide sequence encoding a polypeptide having diacylglycerolacyltransferase activity, wherein the nucleotide sequence is set forthin SEQ ID NO: 133, 134, 146, 161, 175, 214, 233, 264, 271, 298, 303,305, 307, 309, 311, 313, 315, 317, 319, 321, 350, or 362; or

(c) a complement of the nucleotide sequence of (a) or (b) wherein thecomplement and the nucleotide sequence consist of the same number ofnucleotides and are 100% complementary.

The isolated polynucleotide encoding diacylglycerol acyltransferase maybe obtained from one or more oleaginous organism. These oleaginousorganisms can be, but are not limited to, Torulaspora delbrueckii,Pichia anomala, Debaryomyces hansenii, Candida zeylanoides, Lipomycesstarkeyi, Mucor circinelloides, Phaffia rhodozyma, Rhodotorula glutinis,Cryptococcus curvatus, and Mortierella alpina.

Recombinant DNA construct comprising the isolated nucleic acid fragmentencoding diacylglycerol acyltransferase can be operably linked to atleast one regulatory sequence, and can be incorporated into a cell. Thecell may be from an oilseed plant.

In an eleventh embodiment the present invention concerns a method forincreasing the total fatty acid content of an oilseed comprising:

(a) transforming at least one oilseed cell with the above mentionedrecombinant construct;

(b) selecting the transformed oilseed cell(s) of step (a) having anincreased total fatty acid content when compared to the total fatty acidcontent of a non-transgenic, null segregant oilseed.

In a twelfth embodiment the present invention concerns seed products andby-products and progeny obtained from the oilseeds transformed with theabove mentioned recombinant constructs.

-   -   In a final embodiment the present invention concerns fungi, or        microbial oleaginous organisms, comprising a recombinant DNA        construct comprising any isolated nucleic acid fragments        encoding any diacylglycerol acyltransferase of the present        invention. Further, the fungal cell can be, but is not limited        to, Yarrowia, Candida, Rhodotorula, Rhodosporidium,        Cryptococcus, Trichosporon and Lipomyces.

BRIEF DESCRIPTION OF THE DRAWINGS AND SEQUENCE LISTINGS

The invention can be more fully understood from the following detaileddescription and the accompanying drawings and Sequence Listing, whichform a part of this application.

FIG. 1A provides a plasmid map for pFBAIn-YLDGAT 1.

FIG. 1B provides a plasmid map for pFBAIn-YLDGAT2.

FIG. 1C provides a plasmid map for pFBAIn-MOD 1.

FIG. 2A provides a plasmid map for KS352.

FIG. 2B provides a plasmid map for KS332.

FIG. 3A provides a plasmid map for KS349.

FIG. 3B provides a plasmid map for KS362.

FIG. 3C provides a plasmid map for KS364.

FIG. 4A shows the correlation between the oleic acid content and thetotal esterified fatty acid content for somatic embryos generated withKS352.

FIG. 4B provides a strong correlation (R²≧0.59) between the oleic acidcontent and the total esterified fatty acid content for somatic embryosgenerated with KS349.

FIG. 5A shows the correlation between the oleic acid content and thetotal esterified fatty acid content for somatic embryos generated withKS352.

FIG. 5B provides a strong correlation (R²≧0.7881) between the oleic acidcontent and the total esterified fatty acid content for somatic embryosgenerated with KS362. FIG. 5C provides a strong correlation (R²≧0.754)between the oleic acid content and the total esterified fatty acidcontent for somatic embryos generated with KS349 in combination withKS362.

FIG. 5D provides a strong correlation (R²≧0.6791) between the oleic acidcontent and the total esterified fatty acid content for somatic embryosgenerated with KS364. FIG. 6A provides a correlation between the oleicacid content and the oil content for wild-type control soy seed (T1generation).

FIG. 6B provides a correlation (R²≧0.45) between the oleic acid contentand the oil content for transgenic soy seed (T1 generation) generated byco-transformation of plasmids KS349 and KS362.

FIG. 7A provides oil content and seed weight of T1 seed generated byco-transformation of plasmids KS349 and KS362.

FIG. 7B provides oil content and seed weight of T1 seed generated bytransformation of plasmid KS362 alone.

FIG. 8A provides hybridization results from genomic DNA blots.Hybridization probes were a Yarrowia DGAT1-specific probe.

FIG. 8B provides hybridization results from genomic DNA blots.Hybridization probes were a Yarrowia DGAT2-specific probe. In FIG. 8Aand FIG. 8B, genomic DNA was isolated from transgenic soybeans obtainedfrom events AFS4818.1.2, AFS4818.1.3, AFS4818.1.5, AFS48182.6,AFS4818.1.9 (See Example 6). DNA was digested with EcoRI or HindIII andrun out on a gel and blotted to nylon filters [AFS4818.1.2 lanes 1 and2, AFS4818.1.3 lanes 3 and 4, AFS4818.1.5 lanes 5 and 6, AFS48182.6lanes 7 and 8, AFS4818.1.9 lanes 9 and 10, and lanes 11 and 12 arenon-transgenic wild-type DNA also digested with EcoRi and HindIII].

FIG. 9 provides hybridization results from genomic DNA blots. The blotsare similar to those described in FIG. 8 except the DNAs were alldigested with BstXI and the blot was probed with a Yarrowia DGAT2specific probe.

FIG. 10 Yarowia lipolytica (YL) wild-type DGAT2 activity (black bar) isa normalized activity set to 100%. Mortierella alpine (MA), Torulasporadelbrueckii (TD), Debaryomyces hansenii (DH), Lipomyces starkeyi (LS),Rhodottorula glutinis (RG), Phaffia rhodozyma (PR), Pichia anomala (PA),Candida zeylanoides (CZ), Cryptococcus curvatus (CC), and Murcorcircinelloides (MC) DGAT2 constructs were wild-type (checkered bars) orcodon optimized/motif altered (white bars). The results are the averageof oil levels of the top five events from each construct which are thenthen normalized to the average of the top five events for YL DGAT2tested in the same experimental set. YL, MS, and LS DGAT1 constructs areshown in the last three sets. The TD DGAT2b codonoptimized/motif-altered version was not tested (ND). Details of theconstructs and the assay results can be found in the Examples.

The sequence descriptions summarize the Sequences Listing attachedhereto. The Sequence Listing contains one letter codes for nucleotidesequence characters and the single and three letter codes for aminoacids as defined in the IUPAC-IUB standards described in Nucleic AcidsResearch 13:3021-3030 (1985) and in the Biochemical Journal219(2):345-373 (1984).

Summary Of Nucleic Acid And Protein SEQ ID Numbers Protein Nucleic acidSEQ SEQ ID NO. ID NO. Description and Abbreviation (bp) (aa) Yarrowialipolytica DGAT1 gene  1 (1581 bp) Plasmid pYDA1  2 (8325 bp) Plasmidpy75  3 (7518 bp) Plasmid pY75 YLDGAT1: YLDGAT1 inserted  4 into pY75(9109)  Plasmid pRS425  5 (6849 bp) Plasmid pGDP425  6 (7494 bp)Yarrowia lipolytica DGAT2 gene  9  10 (1545 bp) (514 aa) Plasmid pY75YLDGAT2, pY75 with YL  11 DGAT2 inserted (9070 bp) Yarrowia lipolyticaDGAT1 gene variant with  16 NcoI and NotI sites added (1603 bp) Yarrowialipolytica DGAT2 gene variant with  17 NcoI and NotI sites added (1567bp) Plasmid pFBAIN-MOD-1 18 (6991 bp) Plasmid pFBAIN-YLDGAT, pFBAIN withYL  19 DGAT1 inserted (8568 bp) Plasmid pFBAIN-YLDGAT2  20 (8532 bp)Plasmid pKS123  21 (7049 bp) cal a24-4 22 (1098 bp) Plasmid pKR53B  25(8138 bp) Plasmid pKR72  26 (7085 bp) Plasmid pKR85  27 (7085 bp)Plasmid pPCR85  30 (4827 bp) Plasmid pKR91  31 (15114 bp) Plasmid pKR92 32 (13268 bp) Plasmid pKR92 YL DGAT2, pKR92 with the  33 YL DGAT2 geneinserted (19604 bp) Plasmid pKR92 YL DGAT1 YL DGAT2  34 (20082 bp) Soybean glycinin 1 (GY1) gene (Genbank  35 X15121) (3527 bp) Soybean GY1promoter  36 (690 bp) Plasmid pZBL114  39 (6660 bp) Soybean GM GY1(glycinin 1) gene  40 (1437 bp) Synthetic BHL8 (barley high lysine) gene 41 (204 bp) GY1-BHL8 fusion product  42 (1701 bp) Plasmid pZBL133  43(6493 bp) Plasmid pKS238  44 (6472 bp) Plasmid pKS240  45 (6259 bp)Plasmid pKS120  46 (5267 bp) Plasmid pKS242  47 (8643 bp) Plasmid pKS349 48 (8720 bp) Plasmid pKS121/BS  49 (5280 bp) Plasmid pDs-Red inpKS121/BS  50 (5968 bp) Plasmid pKS332  51 (10058 bp)  Plasmid pKS362 52 (11611 bp)  Soybean promoter GM P34  53 (1422 bp) Plasmid pZBL115 56 (7466 bp) Plasmid pJS89  57 (7841 bp) Morteriella alpina delta-6desaturase gene  58 coding sequence (1390 bp) Plasmid pJS93  59 (9223bp) Plasmid pKS127  60 (7472 bp) Plasmid pKS343  61 (7847 bp) PlasmidpKS352  62 (10866 bp)  Plasmid pKS364  63 (12055 bp)  Yarrowialipolytica DGAT1 gene codon  64  65 optimized for soybean (1581 bp) (526aa) Yarrowia lipolytica DGAT2 gene codon  66  67 optimized for soybean(1545 bp) (514 aa) Plasmid pKR1234  68 (8638 bp) Plasmid ppPSgly32  71(3673 bp) Plasmid pKR264  72 (4171 bp) Plasmid pKR1212  73 (6130 bp)Plasmid pKR1235  74 (5764 bp) Plasmid pKR1236 comprising both Yarrowia 75 lipolytica DGAT1 and DGAT2 (11693 bp)  Plasmid pKR1254 comprisingwild-type  78 Yarrowia lipolytica DGAT2 (5079 bp) Plasmid pKR1254_Y326F,pKR1254  81 comprising mutant Y326F Yarrowia lipolytica (5079 bp) DGAT2Yarrowia lipolytica DGAT2 comprising codon  82  83 326 mutated from Tyrto Phe (1545 bp) (514 aa) Plasmid pKR1254_Y326L, pKR1254  86 comprisingmutant Y326L Yarrowia lipolytica (5079 bp) DGAT2 Yarrowia lipolyticaDGAT2 comprising codon  87  88 326 mutated from Tyr to Leu (1545 bp)(514 aa) Plasmid pKR1254_R327K, pKR1254  91 comprising mutant R327KYarrowia lipolytica (5079 bp) DGAT2 Yarrowia lipolytica DGAT2 comprisingcodon  92  93 327 mutated from Arg to Lys (1545 bp) (514 aa) PlasmidpY191 yeast expression vector  94 comprising wild-type Yarrowialipolytica (9074 bp) DGAT2 Plasmid pY192 yeast expression vector  95comprising mutant Y326F Yarrowia lipolytica (9074 bp) DGAT2 PlasmidpY193 yeast expression vector  96 comprising mutant Y326L Yarrowialipolytica (9074 bp) DGAT2 Plasmid pY194 yeast expression vector  97comprising mutant R327K Yarrowia lipolytica (9074 bp) DGAT2 PlasmidpKR1256 soybean expression vector  98 comprising wild-type Yarrowialipolytica (8641 bp) DGAT2 Plasmid pKR1277 soybean expression vector  99comprising mutant Y326F Yarrowia lipolytica (8641 bp) DGAT2 PlasmidpKR1278 soybean expression vector 100 comprising mutant Y326L Yarrowialipolytica (8641 bp) DGAT2 Plasmid pKS392 comprising Yarrowia 101lipolytica DGAT1 codon optimized for soybean (11647 bp)  driven byb-conglycinin promoter Plasmid pKS393 comprising Yarrowia 102 lipolyticaDGAT2 codon optimized for soybean (11611 bp)  driven by b-con promoterPlasmid pKS391 comprising wild-type 103 Yarrowia lipolytica DGAT1 drivenby b-con (11649 bp)  promoter Consensus sequence from 5 independently131 isolated cosmids for Td (Torulaspora (2567 bp) delbrueckii) DGAT2AConsensus sequence from 5 independently 132 isolated cosmids for Td(Torulaspora (2700 bp) delbrueckii) DGAT2B Genomic sequence for Td(Torulaspora 133 135 delbrueckii) DGAT2A (1362 bp) (453 aa) Genomicsequence for Td (Torulaspora 134 136 delbrueckii) DGAT2B (1362 bp) (453aa) Consensus sequence from 5 independently 145 isolated cosmids for Pa(Pichia anomala) (2062 bp) DGAT2 Genomic sequence for Pa (Pichiaanomala) 146 147 DGAT2 (1290 bp) (429 aa) Consensus sequence from 3independently 160 isolated cosmids for Dh (Debaryomyces (2800 bp)hansenii) DGAT2 Genomic sequence for Dh (Debaryomyces 161 162 hansenii)DGAT2 (2028 bp) (675 aa) Consensus sequence from 4 independently 174isolated cosmids for Cz (Candida zeylanoides) (3021 bp) DGAT2 Genomicsequence for Cz (Candida 175 176 zeylanoides) DGAT2 (1695 bp) (564 aa)Consensus sequence from 2 independently 189 isolated cosmids for Ls(Lipomyces starkeyi) (3343 bp) DGAT1 Genomic sequence for Ls (Lipomycesstarkeyi) 194 DGAT2 (2090 bp) Genomic sequence from 7 independently 204isolated cosmids for Rg (Rhodotorula glutinis) (2944 bp) DGAT2 PCR/RACEassembled DGAT2 from Pr 214 215 (Phaffia rhodozyma) (1218 bp) (405 aa)Consensus sequence from 7 independently 226 isolated cosmids for Cc(Cryptococcus (2816 bp) curvatus) PCR/RACE assembled DGAT2 from Mc 233234 (Mucor circinelloides) (1110 bp) (369 aa) Plasmid pKR1295 comprisingthe Torulaspora 237 delbrueckii DGAT2a (4895 bp) Plasmid pKR1324comprising the Torulaspora 238 delbrueckii DGAT2a (8457 bp) PlasmidpKR1296 comprising the Torulaspora 241 delbrueckii DGAT2b (4896 bp)Plasmid pKR1325 comprising the Torulaspora 242 delbrueckii DGAT2b (8458bp) Plasmid pKR1297comprising Debaryomyces 245 hansenii DGAT2 (5561 bp)Plasmid pKR179 comprising Debaryomyces 246 hansenii DGAT2 (4480 bp)Plasmid pKR1327comprising Debaryomyces 247 hansenii DGAT2 (6519 bp)Plasmid pKR325comprising Debaryomyces 248 hansenii DGAT2 (5303 bp)Plasmid pKR1328comprising Debaryomyces 249 hansenii DGAT2 (9122 bp)Plasmid pMDGAT2-17 comprising Mortierella 250 alpina DGAT2 (8084 bp)Plasmid pKR1330 comprising Mortierella 253 alpina DGAT2 (4529 bp)Mortierella alpina DGAT2 254 255 (996 bp) (331 aa) Plasmid pKR1335comprising Mortierella 256 alpina DGAT2 (8091 bp) Plasmid pKR1319comprising Pichia anomala 259 DGAT2 (4824 bp) Plasmid pKR1332 comprisingPichia anomala 260 DGAT2 (8386 bp) Plasmid pHD28 comprising Rhodotorula263 glutinis DGAT2 (4573 bp) Rhodotorula glutinis DGAT2 264 265 (1041bp) (346 aa) Plasmid pKR1333 comprising Rhodotorula 266 glutinis DGAT2(8137 bp) Plasmid pHD30 comprising Lipomyces starkeyi 270 DGAT2 (4766bp) Lipomyces starkeyi DGAT2 271 272 (1233 bp) (410 aa) Plasmid pKR1337comprising Lipomyces 273 starkeyi DGAT2 (8329 bp) Plasmid pMDGAT1-17comprising Mortierella 274 alpina DGAT1 (8666 bp) Plasmid pKR1329comprising Mortierella 277 alpina DGAT1 (5111 bp) Mortierella alpinaDGAT1 278 279 (1578 bp) (525 aa) Plasmid pKR1334 comprising Mortierella280 alpina PL DGAT1 (8674 bp) Plasmid pKR1314 comprising Yarrowia 281lipolytica DGAT1 (4547 bp) Plasmid pKR1310 comprising part of the codon286 optimized Yarrowia lipolytica DGAT1 (4749 bp) Plasmid pKR1316comprising codon optimized 287 Yarrowia lipolytica DGAT1 (5136 bp) Codonoptimized Yarrowia lipolytica DGAT1 288 (1581 bp) Plasmid pKR1323comprising codon optimized 289 Yarrowia lipolytica DGAT1 (8677 bp)Plasmid pHD37 comprising Phaffia rhodozyma 292 DGAT2 (4751 bp) Phaffiarhodozyma DGAT2 293 (1218 bp) Plasmid pKR1372 comprising Phaffia 294rhodozyma DGAT2 (8314 bp) Plasmid pHD38 comprising Lipomyces starkeyi297 DGAT1 (5198 bp) Lipomyces starkeyi DGAT1 298 299 (1665 bp) (554 aa)Plasmid pKR1375 comprising Lipomyces 300 starkeyi DGAT1 (8761 bp) Codonoptimized Mortierella alpina DGAT1 301 (1578 bp) Codon optimizedLipomyces starkeyi DGAT1 302 (1665 bp) Codon optimized and Tyr to Phemutated 303 304 Torulaspora delbrueckii DGAT2A (1362 bp) (453 aa) Codonoptimized and Tyr to Phe mutated 305 306 Torulaspora delbrueckii DGAT2B(1362 bp) (453 aa) Codon optimized and Tyr to Phe mutated 307 308 Pichiaanomala DGAT2 (1290 bp) (429 aa) Codon optimized and Tyr to Phe mutated309 310 Debaryomyces hansenii DGAT2 (2028 bp) (675 aa) Codon optimizedand Tyr to Phe mutated 311 312 Candida zeylanoides DGAT2 (1695 bp) (564aa) Codon optimized and Tyr to Phe mutated 313 314 Lipomyces starkeyiDGAT2 (1233 bp) (410 aa) Codon optimized and Tyr to Phe mutated 315 316Mucor circinelloides DGAT2 (1110 bp) (369 aa) Codon optimized and Tyr toPhe mutated 317 318 Phaffia rhodozyma DGAT2 (1218 bp) (405 aa) Codonoptimized and Tyr to Phe mutated 319 320 Rhodotorula glutinis DGAT2(1041 bp) (346 aa) Codon optimized and Tyr to Phe mutated 321 322Mortierella alpina DGAT2 (1110 bp) (369 aa) plasmid pKR278  323 (5303)plasmid pKR1274  324 (8358) Soybean thioesterase2 gene  325 (1251)plasmid pTC4  326 (9592) plasmid pKR1258  331 (4738) Soybean Fad 2-1gene  332 (1164) plasmid pBS43  333 (10303) plasmid PCRblunt-Fad2-1  338(4584) plasmid pKR1259  339 (5797) plasmid pKR1261  340 (7590) plasmidpKR123R  341 (4993) plasmid pKR1266  342 (9036) plasmid pKR1267  343(11615) plasmid pKR457  344 (5252) plasmid pKR1264  345 (9295) plasmidpKR1277  346 (2577) plasmid pKR1269  347 (9219) Cryptococcus curvatusDGAT2 gene  350 351 (1506) (501) plasmid pHD39  354 (5227) plasmidpKR1392  355 (8791) plasmid pKR1408  358 (4643) Mucor circinelloidesDGAT2 gene  359 (1110) plasmid pKR1409  360 (8206) plasmid pKR1427  361(8602) Cryptococcus curvatus DGAT2 gene optimized  362 363 for soyexpression (1506) (501) plasmid pKR1422  364 (8458) plasmid pKR1421  365(8386) plasmid pKR1420  366 (9124) plasmid pKR1512  367 (8791) plasmidpKR1415  368 (8329) plasmid pKR1513  369 (8206) plasmid pKR1416  370(8314) plasmid pKR1423  371 (8137) plasmid pKR1419  372 (8092) plasmidpKR1522  373 (8602) plasmid pKR1514  374 (8761) plasmid pKR1511  375(8674)

SEQ ID NOs:7-8 correspond to PCR primers oYLDGAT2-1 (SEQ IOD NO:7) andoYLDGAT2-2 (SEQ IOD NO:8), used to amplify the Yarrowia lipolyticadiacylglycerol acyltransferase 2 (YL DGAT2) gene from a yeast lysate(for details see Example 1.)

SEQ ID NOs:12-15 correspond to oligonucleotide primers used to amplifythe coding regions of YL DGAT1 (YDGAT1-F and YDGAT1-R; SEQ ID NOs:12-13,respectively) and YL DGAT2 (YDGAT2-F and YDGAT2-R, SEQ ID NOs:14-15,respectively) from Yarrowia lipolytica genomic DNA.

SEQ ID NOs:23 (oCal-15) and SEQ ID NO:24 (oCal-6) correspond tooligonucleotide primers used to amplify DNA fragment cal a24-4 (SEQ IDNO:22) from template plasmid CalFad2-2 described in PCT Publication No.WO 02/008269.

SEQ ID NO:28 (oKR85-1) and SEQ ID NO:29 (oKR85-2) correspond to primersused to amplify the beta-conglycinin promoter-(NotI cloningsite)-phaseolin 3′ terminator region from plasmid pKR85 (SEQ ID NO:27.)

SEQ ID NOs:37(oGy1-1) and SEQ ID NO:38 (oGy1-2) correspond to primersused to amplify the soybean glycinin 1 promoter (SEQ ID NO:36) andincorporating BamHI and NcoI sites on the 5′ and 3′-ends, respectively.

SEQ ID NO:54 (oP34-1) and SEQ ID NO:55 (oP34-2) correspond to primersused to amplify the soybean P34 promoter (SEQ ID NO:53) andincorporating BamHI and NotI sites into the 5′ and 3′-ends,respectively.

SEQ ID NO:69 (oSGly-2) and SEQ ID NO:70 (oSGly-3) correspond to primersused to amplify the glycinin GY1 promoter.

SEQ ID NOs:76(oYDG2-1) and SEQ ID NO:77 (oYDG2-2) correspond to primersused to amplify Yarrowia DGAT2 (SEQ ID NO:10) which was thenincorporated into pKR1254 (SEQ ID NO:78).

SEQ ID NO:79 (Y1D2_Y326F-5) and SEQ ID NO:80 (Y1D2_Y326F-3) correspondto primers used to mutate the amino acid at position 326 of YarrowiaDGAT2 (SEQ ID NO:10) from tyrosine to phenylalanine.

SEQ ID NO:84 (Y1D2_Y326L-5) and SEQ ID NO:85 (Y1D2_Y326L-3) correspondto primers used to mutate the amino acid at position 326 of YarrowiaDGAT2 (SEQ ID NO:10) from tyrosine to leucine.

SEQ ID NO:89 (Y1D2_R327K-5) and SEQ ID NO:90 (Y1D2_R327K-3) correspondto primers used to mutate the amino acid at position 327 of YarrowiaDGAT2 (SEQ ID NO:10) from arginine to lysine.

SEQ ID NO:104 (MWG619) and SEQ ID NO:105 (MWG620) correspond to primersused to amplify 26S ribosomal DNA sequences.

SEQ ID NO:106 (primer 26S TD) is the 26S rDNA fragment isolated fromTorulaspora delbrueckii. These sequencing fragments were used toidentify the organisms from which the genomic DNA was isolated.

SEQ ID NOs:107-120 are degenerate primers used to amplify DGAT1 andDGAT2 genes from oleaginous organismal genomic DNA. See Table 37 inExample 18 for details.

SEQ ID NO:121 is the PCR fragment isolated from Torulaspora delbrueckiithat was used as a probe to screen cosmid libraries. The translation ofthis fragment is shown in SEQ ID NO:122.

SEQ ID NOs:123-130 are sequencing primers used in characterizing theTorulaspora delbrueckii DGAT2 containing cosmid clones.

SEQ ID NO:137 is the 26S rDNA fragment isolated from Pichia anomala.

SEQ ID NO:138 is the PCR fragment isolated from Pichia anomala that wasused as a probe to screen cosmid libraries. The translation of thisfragment is shown in SEQ ID NO:139.

SEQ ID NOs:140-144 are sequencing primers used in characterizing thePichia anomala DGAT2 containing cosmid clones.

SEQ ID NO:148 is the PCR fragment isolated from Debaryomyces hanseniithat was used as a probe to screen cosmid libraries. The translation ofthis fragment is shown in SEQ ID NO:149.

SEQ ID NOs:150-159 are sequencing primers used in characterizing theDebaryomyces hansenii DGAT2 containing cosmid clones.

SEQ ID NO:163 is the 26S rDNA fragment isolated from Candidazeylanoides. SEQ ID NO:164 is the PCR fragment isolated from Candidazeylanoides that was used as a probe to screen cosmid libraries. Thetranslation of this fragment is shown in SEQ ID NO:165.

SEQ ID NOs:166-173 are sequencing primers used in characterizing theCandida zeylanoides DGAT2 containing cosmid clones.

SEQ ID NO:177 is the PCR fragment isolated from Lipomyces starkeyi thatwas used as a DGAT1 probe to screen cosmid libraries. The translation ofthis fragment is shown in SEQ ID NO:178.

SEQ ID NO:179 is the PCR fragment isolated from Lipomyces starkeyi thatwas used as a DGAT2 probe to screen cosmid libraries. The translation ofthis fragment is shown in SEQ ID NO:180.

SEQ ID NOs:181-188 are sequencing primers used in characterizing theLipomyces starkeyi DGAT1 containing cosmid clones.

SEQ ID NOs:190-193 are sequencing primers used in characterizing theLipomyces starkeyi DGAT2 containing cosmid clones.

SEQ ID NO:195 is the PCR fragment isolated from Rhodotorula glutinisthat was used as a DGAT2 probe to screen cosmid libraries. Thetranslation of this fragment is shown in SEQ ID NO:196.

SEQ ID NOs:197-203 are sequencing primers used in characterizing theRhodotorula glutinis DGAT2 containing cosmid clones.

SEQ ID NO:205 is the PCR fragment isolated from Phaffia rhodozyma thatencodes a partial DGAT2. The translation of this fragment is shown inSEQ ID NO:206.

SEQ ID NOs:207-213 are sequencing primers used in characterizing thePhaffia rhodozyma DGAT2 containing cosmid clones.

SEQ ID NO:216 is the PCR fragment isolated from Cryptococcus curvatusthat was used as a DGAT2 probe to screen cosmid libraries. Thetranslation of this fragment is shown in SEQ ID NO:217.

SEQ ID NOs:218-225 are sequencing primers used in characterizing theCryptococcus curvatus DGAT2 containing cosmid clones.

SEQ ID NO:227 is the PCR fragment isolated from Mucor circinelloidesthat was used as a DGAT2 probe to screen cosmid libraries. Thetranslation of this fragment is shown in SEQ ID NO:228.

SEQ ID NOs:229-232 are sequencing primers used in characterizing theMucor circinelloides DGAT2 containing cosmid clones.

SEQ ID NOs:235-237 are primers used in amplifying the Torulasporadelbrueckii DGAT2a for cloning into plasmid pKR1324.

SEQ ID NOs:239-240 are primers used in amplifying the Torulasporadelbrueckii DGAT2b for cloning into plasmid pKR1325.

SEQ ID NOs:243-244 are primers used in amplifying the Debaryomyceshansenii DGAT2 for cloning into plasmid pKR1327.

SEQ ID NOs:243-244 are primers used in amplifying the Mortierella alpinaDGAT2 for cloning into plasmid pKR1330.

SEQ ID NOs:257-258 are primers used in amplifying the Pichia anomalaDGAT2 for cloning into plasmid pKR1319.

SEQ ID NOs:261-262 are primers used in amplifying the Rhodotorulaglutinis DGAT2 for cloning into plasmid pHD28.

SEQ ID NOs:267-269 are primers used in amplifying the Lipomyces starkeyiDGAT2 for cloning into plasmid pHD30.

SEQ ID NOs:275-276 are primers used in amplifying the Mortierella alpinaDGAT1 for cloning into plasmid pKR1329.

SEQ ID NOs:282-285 are primers used in the codon optimization of theYarrowia lipolytica DGAT1 for cloning into plasmid pKR1310.

SEQ ID NOs:290-291 are primers used in the amplification of the Pharmarhodozyma DGAT2 for cloning into plasmid pHD37.

SEQ ID NOs:295-296 are primers used in amplifying the Lipomyces starkeyiDGAT1 for cloning into plasmid pHD38.

SEQ ID NO:327-330 (GmTE2 5-1, GmTE2 3-1, GmTE2 5-2, and GmTE2 3-2,respectively) correspond to primers used to amplify the soybeanthioesterase 2 gene.

SEQ ID NO:334-337 (GmFad2-1 5-1, GmFad2-1 3-1, GmFad2-1 5-2, andGmFad2-1 3-2, respectively) correspond to primers used to amplify thesoybean fatty acid desaturase 2-1 gene.

SEQ ID NO:348-349 (CC ORF FWD and CC ORF REV, respectively) correspondto primers used to amplify the Cryptococcus curvatus diacylglycerideacyltransferase 2 gene.

SEQ ID NO:352-353 (CzDGAT2-5 and CzDGAT2-3, respectively) correspond toprimers used to amplify the Candida zeylanoides diacylglycerideacyltransferase 2 gene.

SEQ ID NO:356-357 (oMcDG2-1 and oMcDG2-2, respectively) correspond toprimers used to amplify the Mucor circinelloides diacylglycerideacyltransferase 2 gene.

DETAILED DESCRIPTION OF THE INVENTION

The disclosure of each reference set forth herein is hereby incorporatedby reference in its entirety.

As used herein and in the appended claims, the singular forms “a”, “an”,and “the” include plural reference unless the context clearly dictatesotherwise. Thus, for example, reference to “a plant” includes aplurality of such plants, reference to “a cell” includes one or morecells and equivalents thereof known to those skilled in the art, and soforth.

In the context of this disclosure, a number of terms and abbreviationsare used. The following definitions are provided.

“Open reading frame” is abbreviated ORF.

“Polymerase chain reaction” is abbreviated PCR.

“American Type Culture Collection” is abbreviated ATCC.

Acyl-CoA:sterol-acyltransferase” is abbreviated ARE2.

“Phospholipid:diacylglycerol acyltransferase” is abbreviated PDAT.

“Diacylglycerol acyltransferase” is abbreviated DAG AT or DGAT.

“Diacylglycerol” is abbreviated DAG.

“Triacylglycerols” are abbreviated TAGs.

“Co-enzyme A” is abbreviated CoA.

The term “fatty acids” refers to long chain aliphatic acids (alkanoicacids) of varying chain length, from about C₁₂ to C₂₂ (although bothlonger and shorter chain-length acids are known). The predominant chainlengths are between C₁₆ and C₂₂. The structure of a fatty acid isrepresented by a simple notation system of “X:Y”, where X is the totalnumber of carbon (C) atoms in the particular fatty acid and Y is thenumber of double bonds.

Generally, fatty acids are classified as saturated or unsaturated. Theterm “saturated fatty acids” refers to those fatty acids that have no“double bonds” between their carbon backbone. In contrast, “unsaturatedfatty acids” have “double bonds” along their carbon backbones (which aremost commonly in the cis-configuration). “Monounsaturated fatty acids”have only one “double bond” along the carbon backbone (e.g., usuallybetween the 9^(th) and 10^(th) carbon atom as for palmitoleic acid(16:1) and oleic acid (18:1)), while “polyunsaturated fatty acids” (or“PUFAs”) have at least two double bonds along the carbon backbone (e.g.,between the 9^(th) and 10^(th), and 12^(th) and 13^(th) carbon atoms forlinoleic acid (18:2); and between the 9^(th) and 10^(th), 12^(th) and13^(th), and 15^(th), and 16^(th) for α-linolenic acid (18:3)).

“Microbial oils” or “single cell oils” are those oils naturally producedby microorganisms (e.g., algae, oleaginous yeasts and filamentous fungi)during their lifespan. The term “oil” refers to a lipid substance thatis liquid at 25° C. and usually polyunsaturated. In contrast, the term“fat” refers to a lipid substance that is solid at 25° C. and usuallysaturated.

“Lipid bodies” refer to lipid droplets that usually are bounded byspecific proteins and a monolayer of phospholipid. These organelles aresites where most organisms transport/store neutral lipids. Lipid bodiesare thought to arise from microdomains of the endoplasmic reticulum thatcontain TAG-biosynthesis enzymes; and, their synthesis and size appearto be controlled by specific protein components.

“Neutral lipids” refer to those lipids commonly found in cells in lipidbodies as storage fats and oils and are so called because at cellularpH, the lipids bear no charged groups. Generally, they are completelynon-polar with no affinity for water. Neutral lipids generally refer tomono-, di-, and/or triesters of glycerol with fatty acids, also calledmonoacylglycerol, diacylglycerol or TAG, respectively (or collectively,acylglycerols). A hydrolysis reaction must occur to release free fattyacids from acylglycerols.

The terms “triacylglycerol”, “oil” and “TAGs” refer to neutral lipidscomposed of three fatty acyl residues esterified to a glycerol molecule(and such terms will be used interchangeably throughout the presentdisclosure herein). Such oils can contain long chain PUFAs, as well asshorter saturated and unsaturated fatty acids and longer chain saturatedfatty acids. Thus, “oil biosynthesis” generically refers to thesynthesis of TAGs in the cell.

The term “DAG AT” or “DGAT” refers to a diacylglycerol acyltransferase(also known as an acyl-CoA-diacylglycerol acyltransferase or adiacylglycerol O-acyltransferase) (EC 2.3.1.20). This enzyme isresponsible for the conversion of acyl-CoA and 1,2-diacylglycerol to TAGand CoA (thereby involved in the terminal step of TAG biosynthesis). Twofamilies of DAG AT enzymes exist: DGAT1 and DGAT2. The former familyshares homology with the acyl-CoA:cholesterol acyltransferase (ACAT)gene family, while the latter family is unrelated (Lardizabal et al., J.Biol. Chem. 276(42):38862-28869 (2001)).

The term “PDAT” refers to a phospholipid:diacylglycerol acyltransferaseenzyme (EC 2.3.1.158). This enzyme is responsible for the transfer of anacyl group from the sn-2 position of a phospholipid to the sn-3 positionof 1,2-diacylglycerol, thus resulting in lysophospholipid and TAG(thereby involved in the terminal step of TAG biosynthesis).

This enzyme differs from DGAT (EC 2.3.1.20) by synthesizing TAG via anacyl-CoA-independent mechanism.

The term “ARE2” refers to an acyl-CoA:sterol-acyltransferase enzyme (EC2.3.1.26; also known as a sterol-ester synthase 2 enzyme), catalyzingthe following reaction: acyl-CoA+cholesterol=CoA+cholesterol ester.

As used herein, “nucleic acid” means a polynucleotide and includessingle or double-stranded polymer of deoxyribonucleotide orribonucleotide bases. Nucleic acids may also include fragments andmodified nucleotides. Thus, the terms “polynucleotide”, “nucleic acidsequence”, “nucleotide sequence” or “nucleic acid fragment” are usedinterchangeably and is a polymer of RNA or DNA that is single- ordouble-stranded, optionally containing synthetic, non-natural or alterednucleotide bases. Nucleotides (usually found in their 5′-monophosphateform) are referred to by their single letter designation as follows: “A”for adenylate or deoxyadenylate (for RNA or DNA, respectively), “C” forcytidylate or deosycytidylate, “G” for guanylate or deoxyguanylate, “U”for uridlate, “T” for deosythymidylate, “R” for purines (A or G), “Y”for pyrimidiens (C or T), “K” for G or T, “H” for A or C or T, “I” forinosine, and “N” for any nucleotide.

The terms “subfragment that is functionally equivalent” and“functionally equivalent subfragment” are used interchangeably herein.These terms refer to a portion or subsequence of an isolated nucleicacid fragment in which the ability to alter gene expression or produce acertain phenotype is retained whether or not the fragment or subfragmentencodes an active enzyme. For example, the fragment or subfragment canbe used in the design of chimeric genes to produce the desired phenotypein a transformed plant Chimeric genes can be designed for use insuppression by linking a nucleic acid fragment or subfragment thereof,whether or not it encodes an active enzyme, in the sense or antisenseorientation relative to a plant promoter sequence.

The term “conserved domain” or “motif” means a set of amino acidsconserved at specific positions along an aligned sequence ofevolutionarily related proteins. While amino acids at other positionscan vary between homologous proteins, amino acids that are highlyconserved at specific positions indicate amino acids that are essentialin the structure, the stability, or the activity of a protein. Becausethey are identified by their high degree of conservation in alignedsequences of a family of protein homologues, they can be used asidentifiers, or “signatures”, to determine if a protein with a newlydetermined sequence belongs to a previously identified protein family.

The terms “homology”, “homologous”, “substantially similar” and“corresponding substantially” are used interchangeably herein. Theyrefer to nucleic acid fragments wherein changes in one or morenucleotide bases do not affect the ability of the nucleic acid fragmentto mediate gene expression or produce a certain phenotype. These termsalso refer to modifications of the nucleic acid fragments of the instantinvention such as deletion or insertion of one or more nucleotides thatdo not substantially alter the functional properties of the resultingnucleic acid fragment relative to the initial, unmodified fragment. Itis therefore understood, as those skilled in the art will appreciate,that the invention encompasses more than the specific exemplarysequences.

Moreover, the skilled artisan recognizes that substantially similarnucleic acid sequences encompassed by this invention are also defined bytheir ability to hybridize (under moderately stringent conditions, e.g.,0.5×SSC, 0.1% SDS, 60° C.) with the sequences exemplified herein, or toany portion of the nucleotide sequences disclosed herein and which arefunctionally equivalent to any of the nucleic acid sequences disclosedherein. Stringency conditions can be adjusted to screen for moderatelysimilar fragments, such as homologous sequences from distantly relatedorganisms, to highly similar fragments, such as genes that duplicatefunctional enzymes from closely related organisms. Post-hybridizationwashes determine stringency conditions.

The term “selectively hybridizes” includes reference to hybridization,under stringent hybridization conditions, of a nucleic acid sequence toa specified nucleic acid target sequence to a detectably greater degree(e.g., at least 2-fold over background) than its hybridization tonon-target nucleic acid sequences and to the substantial exclusion ofnon-target nucleic acids. Selectively hybridizing sequences typicallyhave about at least 80% sequence identity, or 90% sequence identity, upto and including 100% sequence identity (i.e., fully complementary) witheach other.

The term “stringent conditions” or “stringent hybridization conditions”includes reference to conditions under which a probe will selectivelyhybridize to its target sequence. Stringent conditions aresequence-dependent and will be different in different circumstances. Bycontrolling the stringency of the hybridization and/or washingconditions, target sequences can be identified which are 100%complementary to the probe (homologous probing). Alternatively,stringency conditions can be adjusted to allow some mismatching insequences so that lower degrees of similarity are detected (heterologousprobing). Generally, a probe is less than about 1000 nucleotides inlength, optionally less than 500 nucleotides in length.

Typically, stringent conditions will be those in which the saltconcentration is less than about 1.5 M Na ion, typically about 0.01 to1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and thetemperature is at least about 30° C. for short probes (e.g., 10 to 50nucleotides) and at least about 60° C. for long probes (e.g., greaterthan 50 nucleotides). Stringent conditions may also be achieved with theaddition of destabilizing agents such as formamide. Exemplary lowstringency conditions include hybridization with a buffer solution of 30to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulphate) at 37° C.,and a wash in 1× to 2×SSC (20×SSC=3.0 M NaCl/0.3 M trisodium citrate) at50 to 55° C. Exemplary moderate stringency conditions includehybridization in 40 to 45% formamide, 1 M NaCl, 1% SDS at 37° C., and awash in 0.5× to 1×SSC at 55 to 60° C. Exemplary high stringencyconditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at37° C., and a wash in 0.1×SSC at 60 to 65° C.

Specificity is typically the function of post-hybridization washes, thecritical factors being the ionic strength and temperature of the finalwash solution. For DNA-DNA hybrids, the T_(m) can be approximated fromthe equation of Meinkoth et al., Anal. Biochem. 138:267-284 (1984): T_(m)=81.5° C.+16.6 (log M)+0.41 (% GC)−0.61 (% form)−500/L; where M isthe molarity of monovalent cations, % GC is the percentage of guanosineand cytosine nucleotides in the DNA, % form is the percentage offormamide in the hybridization solution, and L is the length of thehybrid in base pairs. The T_(m) is the temperature (under defined ionicstrength and pH) at which 50% of a complementary target sequencehybridizes to a perfectly matched probe. T_(m) is reduced by about 1° C.for each 1% of mismatching; thus, T_(m), hybridization and/or washconditions can be adjusted to hybridize to sequences of the desiredidentity. For example, if sequences with ≧90% identity are sought, theT_(m) can be decreased 10° C. Generally, stringent conditions areselected to be about 5° C. lower than the thermal melting point (T_(m))for the specific sequence and its complement at a defined ionic strengthand pH. However, severely stringent conditions can utilize ahybridization and/or wash at 1, 2, 3, or 4° C. lower than the thermalmelting point (T_(m)); moderately stringent conditions can utilize ahybridization and/or wash at 6, 7, 8, 9, or 10° C. lower than thethermal melting point (T_(m)); low stringency conditions can utilize ahybridization and/or wash at 11, 12, 13, 14, 15, or 20° C. lower thanthe thermal melting point (T_(m)). Using the equation, hybridization andwash compositions, and desired T_(m), those of ordinary skill willunderstand that variations in the stringency of hybridization and/orwash solutions are inherently described. If the desired degree ofmismatching results in a T_(m) of less than 45° C. (aqueous solution) or32° C. (formamide solution) it is preferred to increase the SSCconcentration so that a higher temperature can be used. An extensiveguide to the hybridization of nucleic acids is found in Tijssen,Laboratory Techniques in Biochemistry and MolecularBiology—Hybridization with Nucleic Acid Probes, Part I, Chapter 2“Overview of principles of hybridization and the strategy of nucleicacid probe assays”, Elsevier, N.Y. (1993); and Current Protocols inMolecular Biology, Chapter 2, Ausubel et al., Eds., Greene Publishingand Wiley-Interscience, New York (1995). Hybridization and/or washconditions can be applied for at least 10, 30, 60, 90, 120, or 240minutes.

“Sequence identity” or “identity” in the context of nucleic acid orpolypeptide sequences refers to the nucleic acid bases or amino acidresidues in two sequences that are the same when aligned for maximumcorrespondence over a specif ied comparison window.

Thus, “percentage of sequence identity” refers to the value determinedby comparing two optimally aligned sequences over a comparison window,wherein the portion of the polynucleotide or polypeptide sequence in thecomparison window may comprise additions or deletions (i.e., gaps) ascompared to the reference sequence (which does not comprise additions ordeletions) for optimal alignment of the two sequences. The percentage iscalculated by determining the number of positions at which the identicalnucleic acid base or amino acid residue occurs in both sequences toyield the number of matched positions, dividing the number of matchedpositions by the total number of positions in the window of comparisonand multiplying the results by 100 to yield the percentage of sequenceidentity. Useful examples of percent sequence identities include, butare not limited to, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%,or any integer percentage from 50% to 100%. These identities can bedetermined using any of the programs described herein.

Sequence alignments and percent identity or similarity calculations maybe determined using a variety of comparison methods designed to detecthomologous sequences including, but not limited to, the MegAlign™program of the LASERGENE bioinformatics computing suite (DNASTAR Inc.,Madison, Wis.). Within the context of this application it will beunderstood that where sequence analysis software is used for analysis,that the results of the analysis will be based on the “default values”of the program referenced, unless otherwise specified. As used herein“default values” will mean any set of values or parameters thatoriginally load with the software when first initialized.

The “Clustal V method of alignment” corresponds to the alignment methodlabeled Clustal V (described by Higgins and Sharp, CABIOS. 5:151-153(1989); Higgins, D. G. et al. (1992) Comput. Appl. Biosci. 8:189-191)and found in the MegAlign™ program of the LASERGENE bioinformaticscomputing suite (DNASTAR Inc., Madison, Wis.). For multiple alignments,the default values correspond to GAP PENALTY=10 and GAP LENGTHPENALTY=10. Default parameters for pairwise alignments and calculationof percent identity of protein sequences using the Clustal method areKTUPLE=1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5. For nucleicacids these parameters are KTUPLE=2, GAP PENALTY=5, WINDOW=4 andDIAGONALS SAVED=4. After alignment of the sequences using the Clustal Vprogram, it is possible to obtain a “percent identity” by viewing the“sequence distances” table in the same program.

“BLASTN method of alignment” is an algorithm provided by the NationalCenter for Biotechnology Information (NCBI) to compare nucleotidesequences using default parameters.

It is well understood by one skilled in the art that many levels ofsequence identity are useful in identifying polypeptides, from otherspecies, wherein such polypeptides have the same or similar function oractivity. Useful examples of percent identities include, but are notlimited to, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, or anyinteger percentage from 50% to 100%. Indeed, any integer amino acididentity from 50% to 100% may be useful in describing the presentinvention, such as 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%,61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%,75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. Also, ofinterest is any full-length or partial complement of this isolatednucleotide fragment.

“Gene” refers to a nucleic acid fragment that expresses a specificprotein, including regulatory sequences preceding (5′ non-codingsequences) and following (3′ non-coding sequences) the coding sequence.“Native gene” refers to a gene as found in nature with its ownregulatory sequences. “Chimeric gene” refers to any gene that is not anative gene, comprising regulatory and coding sequences that are notfound together in nature. Accordingly, a chimeric gene may compriseregulatory sequences and coding sequences that are derived fromdifferent sources, or regulatory sequences and coding sequences derivedfrom the same source, but arranged in a manner different than that foundin nature. A “foreign” gene refers to a gene not normally found in thehost organism, but that is introduced into the host organism by genetransfer. Foreign genes can comprise native genes inserted into anon-native organism, or chimeric genes. A “transgene” is a gene that hasbeen introduced into the genome by a transformation procedure.

The term “genome” as it applies to a plant cells encompasses not onlychromosomal DNA found within the nucleus, but organelle DNA found withinsubcellular components (e.g., mitochondrial, plastid) of the cell.

A “codon-optimized gene” is a gene having its frequency of codon usagedesigned to mimic the frequency of preferred codon usage of the hostcell.

An “allele” is one of several alternative forms of a gene occupying agiven locus on a chromosome. When all the alleles present at a givenlocus on a chromosome are the same that plant is homozygous at thatlocus. If the alleles present at a given locus on a chromosome differthat plant is heterozygous at that locus.

“Coding sequence” refers to a DNA sequence that codes for a specificamino acid sequence. “Regulatory sequences” refer to nucleotidesequences located upstream (5′ non-coding sequences), within, ordownstream (3′ non-coding sequences) of a coding sequence, and whichinfluence the transcription, RNA processing or stability, or translationof the associated coding sequence. Regulatory sequences may include, butare not limited to: promoters, translation leader sequences, introns,polyadenylation recognition sequences, RNA processing sites, effectorbinding sites and stem-loop structures.

“Promoter” refers to a DNA sequence capable of controlling theexpression of a coding sequence or functional RNA. The promoter sequenceconsists of proximal and more distal upstream elements, the latterelements often referred to as enhancers. Accordingly, an “enhancer” is aDNA sequence that can stimulate promoter activity, and may be an innateelement of the promoter or a heterologous element inserted to enhancethe level or tissue-specificity of a promoter. Promoters may be derivedin their entirety from a native gene, or be composed of differentelements derived from different promoters found in nature, or evencomprise synthetic DNA segments. It is understood by those skilled inthe art that different promoters may direct the expression of a gene indifferent tissues or cell types, or at different stages of development,or in response to different environmental conditions. It is furtherrecognized that since in most cases the exact boundaries of regulatorysequences have not been completely defined, DNA fragments of somevariation may have identical promoter activity. Promoters that cause agene to be expressed in most cell types at most times are commonlyreferred to as “constitutive promoters”. New promoters of various typesuseful in plant cells are constantly being discovered; numerous examplesmay be found in the compilation by Okamuro, J. K., and Goldberg, R. B.Biochemistry of Plants 15:1-82 (1989).

“Translation leader sequence” refers to a polynucleotide sequencelocated between the promoter sequence of a gene and the coding sequence.The translation leader sequence is present in the fully processed mRNAupstream of the translation start sequence. The translation leadersequence may affect processing of the primary transcript to mRNA, mRNAstability or translation efficiency. Examples of translation leadersequences have been described (Turner, R. and Foster, G. D., Mol.Biotechnol. 3:225-236 (1995)).

“3′ non-coding sequences”, “transcription terminator” or “terminationsequences” refer to DNA sequences located downstream of a codingsequence and include polyadenylation recognition sequences and othersequences encoding regulatory signals capable of affecting mRNAprocessing or gene expression. The polyadenylation signal is usuallycharacterized by affecting the addition of polyadenylic acid tracts tothe 3′ end of the mRNA precursor. The use of different 3′ non-codingsequences is exemplified by Ingelbrecht, I. L., et al. Plant Cell1:671-680 (1989).

“RNA transcript” refers to the product resulting from RNApolymerase-catalyzed transcription of a DNA sequence. When the RNAtranscript is a perfect complementary copy of the DNA sequence, it isreferred to as the primary transcript. A RNA transcript is referred toas the mature RNA when it is a RNA sequence derived frompost-transcriptional processing of the primary transcript. “MessengerRNA” or “mRNA” refers to the RNA that is without introns and that can betranslated into protein by the cell. “cDNA” refers to a DNA that iscomplementary to, and synthesized from, a mRNA template using the enzymereverse transcriptase. The cDNA can be single-stranded or converted intodouble-stranded form using the Klenow fragment of DNA polymerase I.“Sense” RNA refers to RNA transcript that includes the mRNA and can betranslated into protein within a cell or in vitro. “Antisense RNA”refers to an RNA transcript that is complementary to all or part of atarget primary transcript or mRNA, and that blocks the expression of atarget gene (U.S. Pat. No. 5,107,065). The complementarity of anantisense RNA may be with any part of the specific gene transcript,i.e., at the 5′ non-coding sequence, 3′ non-coding sequence, introns, orthe coding sequence. “Functional RNA” refers to antisense RNA, ribozymeRNA, or other RNA that may not be translated but yet has an effect oncellular processes. The terms “complement” and “reverse complement” areused interchangeably herein with respect to mRNA transcripts, and aremeant to define the antisense RNA of the message.

The term “operably linked” refers to the association of nucleic acidsequences on a single nucleic acid fragment so that the function of oneis regulated by the other. For example, a promoter is operably linkedwith a coding sequence when it is capable of regulating the expressionof that coding sequence (i.e., the coding sequence is under thetranscriptional control of the promoter). Coding sequences can beoperably linked to regulatory sequences in a sense or antisenseorientation. In another example, the complementary RNA regions of theinvention can be operably linked, either directly or indirectly, 5′ tothe target mRNA, or 3′ to the target mRNA, or within the target mRNA, ora first complementary region is 5′ and its complement is 3′ to thetarget mRNA.

Standard recombinant DNA and molecular cloning techniques used hereinare well known in the art and are described more fully in Sambrook, J.,Fritsch, E. F. and Maniatis, T. Molecular Cloning: A Laboratory Manual;Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y. (1989).Transformation methods are well known to those skilled in the art andare described infra.

“PCR” or “polymerase chain reaction” is a technique for the synthesis oflarge quantities of specific DNA segments and consists of a series ofrepetitive cycles (Perkin Elmer Cetus Instruments, Norwalk, Conn.).Typically, the double-stranded DNA is heat denatured, the two primerscomplementary to the 3′ boundaries of the target segment are annealed atlow temperature and then extended at an intermediate temperature. Oneset of these three consecutive steps is referred to as a “cycle”.

The term “recombinant” refers to an artificial combination of twootherwise separated segments of sequence, e.g., by chemical synthesis orby the manipulation of isolated segments of nucleic acids by geneticengineering techniques.

The terms “plasmid”, “vector” and “cassette” refer to an extrachromosomal element often carrying genes that are not part of thecentral metabolism of the cell, and usually in the form of circulardouble-stranded DNA fragments. Such elements may be autonomouslyreplicating sequences, genome integrating sequences, phage or nucleotidesequences, linear or circular, of a single- or double-stranded DNA orRNA, derived from any source, in which a number of nucleotide sequenceshave been joined or recombined into a unique construction which iscapable of introducing a promoter fragment and DNA sequence for aselected gene product along with appropriate 3′ untranslated sequenceinto a cell. “Transformation cassette” refers to a specific vectorcontaining a foreign gene and having elements in addition to the foreigngene that facilitates transformation of a particular host cell.“Expression cassette” refers to a specific vector containing a foreigngene and having elements in addition to the foreign gene that allow forenhanced expression of that gene in a foreign host (i.e., to a discretenucleic acid fragment into which a nucleic acid sequence or fragment canbe moved.)

The terms “recombinant construct”, “expression construct”, “chimericconstruct”, “construct”, and “recombinant DNA construct” are usedinterchangeably herein. A recombinant construct comprises an artificialcombination of nucleic acid fragments, e.g., regulatory and codingsequences that are not found together in nature. For example, a chimericconstruct may comprise regulatory sequences and coding sequences thatare derived from different sources, or regulatory sequences and codingsequences derived from the same source, but arranged in a mannerdifferent than that found in nature. Such a construct may be used byitself or may be used in conjunction with a vector. If a vector is used,then the choice of vector is dependent upon the method that will be usedto transform host cells as is well known to those skilled in the art.For example, a plasmid vector can be used. The skilled artisan is wellaware of the genetic elements that must be present on the vector inorder to successfully transform, select and propagate host cellscomprising any of the isolated nucleic acid fragments of the invention.The skilled artisan will also recognize that different independenttransformation events will result in different levels and patterns ofexpression (Jones et al., EMBO J. 4:2411-2418 (1985); De Almeida et al.,Mol. Gen. Genetics 218:78-86 (1989)), and thus that multiple events mustbe screened in order to obtain lines displaying the desired expressionlevel and pattern. Such screening may be accomplished by Southernanalysis of DNA, Northern analysis of mRNA expression, immunoblottinganalysis of protein expression, or phenotypic analysis, among others.

The term “expression”, as used herein, refers to the production of afunctional end-product (e.g., a mRNA or a protein [either precursor ormature]).

The term “introduced” means providing a nucleic acid (e.g., expressionconstruct) or protein into a cell. Introduced includes reference to theincorporation of a nucleic acid into a eukaryotic or prokaryotic cellwhere the nucleic acid may be incorporated into the genome of the cell,and includes reference to the transient provision of a nucleic acid orprotein to the cell. Introduced includes reference to stable ortransient transformation methods, as well as sexually crossing. Thus,“introduced” in the context of inserting a nucleic acid fragment (e.g.,a recombinant DNA construct/expression construct) into a cell, means“transfection” or “transformation” or “transduction” and includesreference to the incorporation of a nucleic acid fragment into aeukaryotic or prokaryotic cell where the nucleic acid fragment may beincorporated into the genome of the cell (e.g., chromosome, plasmid,plastid or mitochondrial DNA), converted into an autonomous replicon, ortransiently expressed (e.g., transfected mRNA).

“Mature” protein refers to a post-translationally processed polypeptide(i.e., one from which any pre- or propeptides present in the primarytranslation product have been removed). “Precursor” protein refers tothe primary product of translation of mRNA (i.e., with pre- andpropeptides still present). Pre- and propeptides may be but are notlimited to intracellular localization signals.

“Stable transformation” refers to the transfer of a nucleic acidfragment into a genome of a host organism, including both nuclear andorganellar genomes, resulting in genetically stable inheritance. Incontrast, “transient transformation” refers to the transfer of a nucleicacid fragment into the nucleus, or DNA-containing organelle, of a hostorganism resulting in gene expression without integration or stableinheritance. Host organisms containing the transformed nucleic acidfragments are referred to as “transgenic” organisms.

As used herein, “transgenic” refers to a plant or a cell which compriseswithin its genome a heterologous polynucleotide. Preferably, theheterologous polynucleotide is stably integrated within the genome suchthat the polynucleotide is passed on to successive generations. Theheterologous polynucleotide may be integrated into the genome alone oras part of an expression construct. Transgenic is used herein to includeany cell, cell line, callus, tissue, plant part or plant, the genotypeof which has been altered by the presence of heterologous nucleic acidincluding those transgenics initially so altered as well as thosecreated by sexual crosses or asexual propagation from the initialtransgenic. The term “transgenic” as used herein does not encompass thealteration of the genome (chromosomal or extra-chromosomal) byconventional plant breeding methods or by naturally occurring eventssuch as random cross-fertilization, non-recombinant viral infection,non-recombinant bacterial transformation, non-recombinant transposition,or spontaneous mutation.

“Antisense inhibition” refers to the production of antisense RNAtranscripts capable of suppressing the expression of the target protein.“Co-suppression” refers to the production of sense RNA transcriptscapable of suppressing the expression of identical or substantiallysimilar foreign or endogenous genes (U.S. Pat. No. 5,231,020).Co-suppression constructs in plants previously have been designed byfocusing on overexpression of a nucleic acid sequence having homology toan endogenous mRNA, in the sense orientation, which results in thereduction of all RNA having homology to the overexpressed sequence(Vaucheret et al., Plant J. 16:651-659 (1998); Gura, Nature 404:804-808(2000)). The overall efficiency of this phenomenon is low, and theextent of the RNA reduction is widely variable. More recent work hasdescribed the use of “hairpin” structures that incorporate all, or part,of an mRNA encoding sequence in a complementary orientation that resultsin a potential “stem-loop” structure for the expressed RNA (PCTPublication No. WO 99/53050, published Oct. 21, 1999; PCT PublicationNo. WO 02/00904, published Jan. 3, 2002). This increases the frequencyof co-suppression in the recovered transgenic plants. Another variationdescribes the use of plant viral sequences to direct the suppression, or“silencing”, of proximal mRNA encoding sequences (PCT Publication No. WO98/36083, published Aug. 20, 1998). Both of these co-suppressingphenomena have not been elucidated mechanistically, although geneticevidence has begun to unravel this complex situation (Elmayan et al.,Plant Cell 10:1747-1757 (1998)).

The term “oleaginous” refers to those organisms that tend to store theirenergy source in the form of lipid (Weete, In: Fungal LipidBiochemistry, 2nd Ed., Plenum, 1980). A class of plants identified asoleaginous are commonly referred to as “oilseed” plants. Examples ofoilseed plants include, but are not limited to: soybean (Glycine andSoja sp.), flax (Linum sp.), rapeseed (Brassica sp.), maize, cotton,safflower (Carthamus sp.) and sunflower (Helianthus sp.).

Within oleaginous microorganisms the cellular oil or TAG contentgenerally follows a sigmoid curve, wherein the concentration of lipidincreases until it reaches a maximum at the late logarithmic or earlystationary growth phase and then gradually decreases during the latestationary and death phases (Yongmanitchai and Ward, Appl. Environ.Microbiol. 57:419-25 (1991)).

Also described herein are oleaginous microbial organisms produced by themethods described herein. This therefore includes oleaginous bacteria,algae, moss, euglenoids, stramenopiles fungi and yeast, comprising intheir genome a recombinant construct incorporating an isolated nucleicacid of the present invention. Additionally, lipids and oils obtainedfrom these oleaginous organisms, products obtained from the processingof the lipids and oil, use of these lipids and oil in foods, animalfeeds or industrial applications and/or use of the by-products in foodsor animal feeds are also described. Oleaginous microalgae also existwith the capability of producing oil contents of 20 to 50% of theirtotal dry weight, especially under stress conditions (Hu et al, 2008,Plant J 54:621-639). Examples of microalgae include, but are not limitedto Rhodomonas salina, Crypthecodinium cohnii, Chaetoceros lauderi,Pavlova pinguis, and Emiliania huxleyi. There is currently greatinterest in using oleaginous microalgae to produce oil for biofuels, orfor use as nutraceuticals or cosmetics (Hu et al, 2008, Plant J54:621-639; Waltz, 2009, Nature Biotechnology 27: 15-18.) The approachof overexpressing genes in microalgae to improve oil production forbiofuels applications is being explored (Waltz, 2009, NatureBiotchnology 27:15-18.)

The term “oleaginous yeast” refers to those microorganisms classified asyeasts that make oil. It is not uncommon for oleaginous microorganismsto accumulate in excess of about 25% of their dry cell weight as oil.Examples of oleaginous yeast include, but are no means limited to, thefollowing genera: Yarrowia, Candida, Rhodotorula, Rhodosporidium,Cryptococcus, Trichosporon and Lipomyces.

The term “plant” refers to whole plants, plant organs, plant tissues,seeds, plant cells, seeds and progeny of the same. Plant cells include,without limitation, cells from seeds, suspension cultures, embryos,meristematic regions, callus tissue, leaves, roots, shoots,gametophytes, sporophytes, pollen and microspores.

“Progeny” comprises any subsequent generation of a plant.

“Non-transgenic, null segregant soybean seed” refers to a near isogenicplant or seed that lacks the transgene, and/or a parental plant used inthe transformation process to obtain the transgenic event. Nullsegregants can be plants or seed that do not contain the transgenictrait due to normal genetic segregation during propagation of theheterozygous transgenic plants.

A “kernel” is the corn caryopsis, consisting of a mature embryo andendosperm which are products of double fertilization. The term “corn” or“maize” represents any variety, cultivar, or population of Zea mays L.

“Grain” comprises mature corn kernels produced by commercial growers foron farm use or for sale to customers in both cases for purposes otherthan growing or reproducing the species. The “seed” is the mature cornkernel produced for the purpose of propagating the species and for saleto commercial growers. As used herein the terms seeds, kernels, andgrains can be used interchangeably. The “embryo” or also termed “germ”is a young sporophytic plant, before the start of a period of rapidgrowth (seed germination). The embryo (germ) of corn contains the vastmajority of the oil found in the kernel. The structure of embryo incereal grain includes the embryonic axis and the scutellum. The“scutellum” is the single cotyledon of a cereal grain embryo,specialized for absorption of the endosperm. The “aleurone” is aproteinaceous material, usually in the form of small granules, occurringin the outermost cell layer of the endosperm of corn and other grains.

The present invention concerns a transgenic soybean seed havingincreased total fatty acid content of at least 10% when compared to thetotal fatty acid content of a non-transgenic, null segregant soybeanseed. It is understood that any measurable increase in the total fattyacid content of a transgenic versus a non-transgenic, null segregantwould be useful. Such increases in the total fatty acid content wouldinclude, but are not limited to, at least 1%, 2%, 3%, 4%, 5%, 6%, 7%,8%, 9%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%.

A transgenic oilseed of the invention can comprise a recombinantconstruct having at least one DGAT sequence. This DGAT sequence can beselected from the group consisting of DGAT1, DGAT2 and DGAT1 incombination with DGAT2. Furthermore, at least one DGAT sequence can befrom Yarrowia. Examples of suitable DGAT sequences that can be used topractice the invention are discussed in the Examples below. There can bementioned SEQ ID NOs: 133, 134, 146, 161, 175, 214, 233, 264, 271, 278,298, 301, 303, 305, 307, 309, 311, 313, 315, 317, 319, 321, 350, 352, or362 in the present invention. Those skilled in the art will appreciatethat the instant invention includes, but is not limited to, the DGATsequences disclosed herein.

Such a recombinant construct promoter would comprise differentcomponents such as a promoter which is a DNA sequence that directscellular machinery of a plant to produce RNA from the contiguous codingsequence downstream (3′) of the promoter. The promoter region influencesthe rate, developmental stage, and cell type in which the RNA transcriptof the gene is made. The RNA transcript is processed to produce mRNAwhich serves as a template for translation of the RNA sequence into theamino acid sequence of the encoded polypeptide. The 5′ non-translatedleader sequence is a region of the mRNA upstream of the protein codingregion that may play a role in initiation and translation of the mRNA.The 3′ transcription termination/polyadenylation signal is anon-translated region downstream of the protein coding region thatfunctions in the plant cell to cause termination of the RNA transcriptand the addition of polyadenylate nucleotides to the 3′ end of the RNA.

The origin of the promoter chosen to drive expression of the DGAT codingsequence is not important as long as it has sufficient transcriptionalactivity to accomplish the invention by expressing translatable mRNA forthe desired nucleic acid fragments in the desired host tissue at theright time. Either heterologous or non-heterologous (i.e., endogenous)promoters can be used to practice the invention. For example, suitablepromoters include, but are not limited to: the alpha prime subunit ofbeta conglycinin promoter, the Kunitz trypsin inhibitor 3 promoter, theannexin promoter, the glycinin Gy1 promoter, the beta subunit of betaconglycinin promoter, the P34/Gly Bd m 30K promoter, the albuminpromoter, the Leg A1 promoter and the Leg A2 promoter.

The annexin, or P34, promoter is described in PCT Publication No. WO2004/071178 (published Aug. 26, 2004). The level of activity of theannexin promoter is comparable to that of many known strong promoters,such as: (1) the CaMV 35S promoter (Atanassova et al., Plant Mol. Biol.37:275-285 (1998); Battraw and Hall, Plant Mol. Biol. 15:527-538 (1990);Holtorf et al., Plant Mol. Biol. 29:637-646 (1995); Jefferson et al.,EMBO J. 6:3901-3907 (1987); Wilmink et al., Plant Mol. Biol. 28:949-955(1995)); (2) the Arabidopsis oleosin promoters (Plant et al., Plant Mol.Biol. 25:193-205 (1994); Li, Texas A&M University Ph.D. dissertation,pp. 107-128 (1997)); (3) the Arabidopsis ubiquitin extension proteinpromoters (Callis et al., J Biol. Chem. 265(21):12486-93 (1990)); (4) atomato ubiquitin gene promoter (Rollfinke et al., Gene. 211(2):267-76(1998)); (5) a soybean heat shock protein promoter (Schoffl et al., MolGen Genet. 217(2-3):246-53 (1989)); and, (6) a maize H3 histone genepromoter (Atanassova et al., Plant Mol. Biol. 37(2):275-85 (1989)).

Another useful feature of the annexin promoter is its expression profilein developing seeds. The annexin promoter is most active in developingseeds at early stages (before 10 days after pollination) and is largelyquiescent in later stages. The expression profile of the annexinpromoter is different from that of many seed-specific promoters, e.g.,seed storage protein promoters, which often provide highest activity inlater stages of development (Chen et al., Dev. Genet. 10:112-122 (1989);Ellerstrom et al., Plant Mol. Biol. 32:1019-1027 (1996); Keddie et al.,Plant Mol. Biol. 24:327-340 (1994); Plant et al., (supra); Li, (supra)).The annexin promoter has a more conventional expression profile butremains distinct from other known seed specific promoters. Thus, theannexin promoter will be a very attractive candidate whenoverexpression, or suppression, of a gene in embryos is desired at anearly developing stage. For example, it may be desirable to overexpressa gene regulating early embryo development or a gene involved in themetabolism prior to seed maturation.

Following identification of an appropriate promoter suitable forexpression of a specific DGAT-coding sequence, the promoter is thenoperably linked in a sense orientation using conventional means wellknown to those skilled in the art.

Standard recombinant DNA and molecular cloning techniques used hereinare well known in the art and are described more fully in Sambrook, J.et al., In Molecular Cloning: A Laboratory Manual; 2nd ed.; Cold SpringHarbor Laboratory Press: Cold Spring Harbor, New York, 1989 (hereinafter“Sambrook et al., 1989”) or Ausubel, F. M., Brent, R., Kingston, R. E.,Moore, D. D., Seidman, J. G., Smith, J. A. and Struhl, K., Eds.; InCurrent Protocols in Molecular Biology; John Wiley and Sons: New York,1990 (hereinafter “Ausubel et al., 1990”).

In another aspect, this invention concerns a method method forincreasing the total fatty acid content of a soybean seed comprising:

(a) transforming at least one soybean cell with a recombinant constructhaving at least one DGAT sequence;

(b) selecting the transformed soybean cell(s) of step (a) having anincreased total fatty acid content of at least 10% when compared to thetotal fatty acid content of a non-transgenic, null segregant soybeanseed.

Once the recombinant construct has been made, it may then be introducedinto a plant cell of choice by methods well known to those of ordinaryskill in the art (e.g., transfection, transformation andelectroporation). Oilseed plant cells are the preferred plant cells. Thetransformed plant cell is then cultured and regenerated under suitableconditions permitting selection of those transformed soybean cell(s)having an increased total fatty acid content of at least 10% whencompared to the total fatty acid content of a non-transgenic, nullsegregant soybean seed.

Such recombinant constructs may be introduced into one plant cell; or,alternatively, each construct may be introduced into separate plantcells. Expression in a plant cell may be accomplished in a transient orstable fashion as is described above.

Also within the scope of this invention are seeds or plant partsobtained from such transformed plants.

Plant parts include differentiated and undifferentiated tissuesincluding, but not limited to the following: roots, stems, shoots,leaves, pollen, seeds, tumor tissue and various forms of cells andculture (e.g., single cells, protoplasts, embryos and callus tissue).The plant tissue may be in plant or in a plant organ, tissue or cellculture.

The term “plant organ” refers to plant tissue or a group of tissues thatconstitute a morphologically and functionally distinct part of a plant.The term “genome” refers to the following: (1) the entire complement ofgenetic material (genes and non-coding sequences) that is present ineach cell of an organism, or virus or organelle; and/or (2) a completeset of chromosomes inherited as a (haploid) unit from one parent.

Methods for transforming dicots (primarily by use of Agrobacteriumtumefaciens) and obtaining transgenic plants have been published, amongothers, for: cotton (U.S. Pat. No. 5,004,863; U.S. Pat. No. 5,159,135);soybean (U.S. Pat. No. 5,569,834; U.S. Pat. No. 5,416,011); Brassica(U.S. Pat. No. 5,463,174); peanut (Cheng et al. Plant Cell Rep.15:653-657 (1996); McKently et al. Plant Cell Rep. 14:699-703 (1995));papaya (Ling, K. et al. Bio/technology 9:752-758 (1991)); and pea (Grantet al. Plant Cell Rep. 15:254-258 (1995)). For a review of othercommonly used methods of plant transformation see Newell, C. A. (Mol.Biotechnol. 16:53-65 (2000)). One of these methods of transformationuses Agrobacterium rhizogenes (Tepfler, M. and Casse-Delbart, F.Microbiol. Sci. 4:24-28 (1987)). Transformation of soybeans using directdelivery of DNA has been published using PEG fusion (PCT Publication No.WO 92/17598), electroporation (Chowrira, G. M. et al., Mol. Biotechnol.3:17-23 (1995); Christou, P. et al., Proc. Natl. Acad. Sci. U.S.A.84:3962-3966 (1987)), microinjection and particle bombardement (McCabe,D.E. et. al., Bio/Technology 6:923 (1988); Christou et al., PlantPhysiol. 87:671-674 (1988)).

There are a variety of methods for the regeneration of plants from planttissue. The particular method of regeneration will depend on thestarting plant tissue and the particular plant species to beregenerated. The regeneration, development and cultivation of plantsfrom single plant protoplast transformants or from various transformedexplants is well known in the art (Weissbach and Weissbach, In: Methodsfor Plant Molecular Biology, (Eds.), Academic: San Diego, Calif.(1988)). This regeneration and growth process typically includes thesteps of selection of transformed cells and culturing thoseindividualized cells through the usual stages of embryonic developmentthrough the rooted plantlet stage. Transgenic embryos and seeds aresimilarly regenerated. The resulting transgenic rooted shoots arethereafter planted in an appropriate plant growth medium such as soil.Preferably, the regenerated plants are self-pollinated to providehomozygous transgenic plants. Otherwise, pollen obtained from theregenerated plants is crossed to seed-grown plants of agronomicallyimportant lines. Conversely, pollen from plants of these important linesis used to pollinate regenerated plants. A transgenic plant of thepresent invention containing a desired polypeptide is cultivated usingmethods well known to one skilled in the art.

In addition to the above discussed procedures, practitioners arefamiliar with the standard resource materials which describe specificconditions and procedures for: the construction, manipulation andisolation of macromolecules (e.g., DNA molecules, plasmids, etc.); thegeneration of recombinant DNA fragments and recombinant expressionconstructs; and, the screening and isolating of clones. See, forexample: Sambrook et al., Molecular Cloning: A Laboratory Manual, ColdSpring Harbor: NY (1989); Maliga et al., Methods in Plant MolecularBiology, Cold Spring Harbor: NY (1995); Birren et al., Genome Analysis:Detecting Genes, Vol. 1, Cold Spring Harbor: NY (1998); Birren et al.,Genome Analysis: Analyzing DNA, Vol. 2, Cold Spring Harbor: NY (1998);Plant Molecular Biology: A Laboratory Manual, eds. Clark, Springer: NY(1997).

Examples of oilseed plants include, but are not limited to: soybean,Brassica species, sunflower, maize, cotton, flax and safflower.

In another aspect, this invention concerns a a transgenic corn kernelhaving increased total fatty acid content of at least 10% when comparedto the total fatty acid content of a non-transgenic, null segregant cornkernel. Such a transgenic corn kernel can comprise a recombinantconstruct having at least one DGAT sequence. This DGAT sequence can beselected from the group consisting of DGAT1, DGAT2, or DGAT1 incombination with DGAT2.

In still another aspect, the present invention concerns a method forincreasing the total fatty acid content of a corn kernel comprising:

(a) transforming at least one corn kernel with a recombinant constructhaving at least one DGAT sequence;

(b) selecting the transformed corn kernel(s) of step (a) having anincreased total fatty acid content of at least 10% when compared to thetotal fatty acid content of a non-transgenic, null segregant cornkernel.

The present invention also concerns a transgenic soybean seed havingincreased total fatty acid content of at least 10% and an increasedoleic acid content of at least 25% when compared to the total fatty acidcontent and oleic acid content of a non-transgenic, null segregantsoybean seed. And the present invention further concerns a transgenicsoybean having increased total fatty acid content of at least 10% and atleast any one of i) an increased oleic acid content of at least 25%; ii)a decreased linolenic acid content of at least 25%; iii) a decreasedlinoleic acid content of at least 4%; iv) a decreased palmitic acidcontent of at least 8%; and v) an increased stearic acid content of atlease 14% when compared to the total fatty acid content and oleic,linolenic acid, linoleic acid, palmitic acid or stearic acid,respectively, content of a non-transgenic, null segregant soybean seed.

In still a further aspect, the present invention also concerns a methodfor increasing the total fatty acid content and oleic acid content of asoybean seed comprising:

(a) transforming at least one soybean cell with a recombinant constructhaving at least one DGAT sequence;

(b) selecting the transformed soybean cell(s) of step (a) having anincreased total fatty acid content of at least 10% and an increasedoleic acid content of at least 25% when compared to the total fatty acidcontent and oleic acid content of a non-transgenic, null segregantsoybean seed.

In still yet a further aspect, the present invention concerns a methodfor increasing the total fatty acid content and decreasing linolenicacid content of a soybean seed comprising:

(a) transforming at least one soybean cell with a recombinant constructhaving at least one DGAT sequence;

(b) selecting the transformed soybean cell(s) of step (a) having anincreased total fatty acid content of at least 10% and a decreasedlinolenic acid content of at least 25% when compared to the total fattyacid content and oleic acid content of a non-transgenic, null segregantsoybean seed.

Yet again in a further aspect, the present invention concerns a methodfor increasing the total fatty acid content and decreasing linoleic acidcontent of a soybean seed comprising:

(a) transforming at least one soybean cell with a recombinant constructhaving at least one DGAT sequence;

(b) selecting the transformed soybean cell(s) of step (a) having anincreased total fatty acid content of at least 10% and a decreasedlinoleic acid content of at least 4% when compared to the total fattyacid content and oleic acid content of a non-transgenic, null segregantsoybean seed.

Again in a further aspect, the present invention concerns a method forincreasing the total fatty acid content and decreased palmitic acidcontent of a soybean seed comprising:

(a) transforming at least one soybean cell with a recombinant constructhaving at least one DGAT sequence;

(b) selecting the transformed soybean cell(s) of step (a) having anincreased total fatty acid content of at least 10% and a decreasedpalmitic acid content of at least 8% when compared to the total fattyacid content and oleic acid content of a non-transgenic, null segregantsoybean seed.

In yet another aspect, the present invention concerns a method forincreasing the total fatty acid content and stearic acid content of asoybean seed comprising:

(a) transforming at least one soybean cell with a recombinant constructhaving at least one DGAT sequence;

(b) selecting the transformed soybean cell(s) of step (a) having anincreased total fatty acid content of at least 10% and an increasedstearic acid content of at least 14% when compared to the total fattyacid content and oleic acid content of a non-transgenic, null segregantsoybean seed.

As was discussed above, any of the transgenic oilseeds discussed hereincan comprise a recombinant construct having at least one DGAT sequence.This DGAT sequence can be selected from the group consisting of DGAT1,DGAT2, or DGAT1 in combination with DGAT2. Furthermore, at least oneDGAT sequence is from Yarrowia.

Transformation of monocotyledons using electroporation, particlebombardment, and Agrobacterium have been reported. Transformation andplant regeneration have been achieved in asparagus (Bytebier et al.,Proc. Natl. Acad. Sci. (USA) 84:5354, (1987)); barley (Wan and Lemaux,Plant Physiol 104:37 (1994)); Zea mays (Rhodes et al., Science 240:204(1988), Gordon-Kamm et al., Plant Cell 2:603-618 (1990), Fromm et al.,Bio/Technology 8:833 (1990), Koziel et al., Bio/Technology 11: 194,(1993), Armstrong et al., Crop Science 35:550-557 (1995)); oat (Somerset al., Bio/Technology 10: 15 89 (1992)); orchard grass (Horn et al.,Plant Cell Rep. 7:469 (1988)); rice (Toriyama et al., TheorAppl. Genet.205:34, (1986); Part et al., Plant Mol. Biol. 32:1135-1148, (1996);Abedinia et al., Aust. J. Plant Physiol. 24:133-141 (1997); Zhang andWu, Theor. Appl. Genet. 76:835 (1988); Zhang et al. Plant Cell Rep.7:379, (1988); Battraw and Hall, Plant Sci. 86:191-202 (1992); Christouet al., Bio/Technology 9:957 (1991)); rye (De la Pena et al., Nature325:274 (1987)); sugarcane (Bower and Birch, Plant J. 2:409 (1992));tall fescue (Wang et al., Bio/Technology 10:691 (1992)), and wheat(Vasil et al., Bio/Technology 10:667 (1992); U.S. Pat. No. 5,631,152).

Assays for gene expression based on the transient expression of clonednucleic acid constructs have been developed by introducing the nucleicacid molecules into plant cells by polyethylene glycol treatment,electroporation, or particle bombardment (Marcotte et al., Nature335:454-457 (1988); Marcotte et al., Plant Cell 1:523-532 (1989);McCarty et al., Cell 66:895-905 (1991); Hattori et al., Genes Dev.6:609-618 (1992); Goff et al., EMBO J. 9:2517-2522 (1990)).

Transient expression systems may be used to functionally dissect geneconstructs (see generally, Maliga et al., Methods in Plant MolecularBiology, Cold Spring Harbor Press (1995)). It is understood that any ofthe nucleic acid molecules of the present invention can be introducedinto a plant cell in a permanent or transient manner in combination withother genetic elements such as vectors, promoters, enhancers etc.

In addition to the above discussed procedures, practitioners arefamiliar with the standard resource materials which describe specificconditions and procedures for the construction, manipulation andisolation of macromolecules (e.g., DNA molecules, plasmids, etc.),generation of recombinant organisms and the screening and isolating ofclones, (see for example, Sambrook et al., Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Press (1989); Maliga et al.,Methods in Plant Molecular Biology, Cold Spring Harbor Press (1995);Birren et al., Genome Analysis: Detecting Genes, 1, Cold Spring Harbor,N.Y. (1998); Birren et al., Genome Analysis: Analyzing DNA, 2, ColdSpring Harbor, N.Y. (1998); Plant Molecular Biology: A LaboratoryManual, eds. Clark, Springer, N.Y. (1997)).

The transgenic oilseeds of the invention can be processed to yield oil,protein products and/or by-products that are derivatives obtained byprocessing that have commercial value. One example, of many, useful forillustrating this point are transgenic soybean seeds of the inventionwhich can be processed to yield soy oil, soy products and/or soyby-products.

“Soy products” can include, but are not limited to, those items listedin Table 1A.

TABLE 1A Soy Protein Products Derived from Soybean Seeds^(a) WholeSoybean Products Roasted Soybeans Baked Soybeans Soy Sprouts Soy MilkSpecialty Soy Foods/Ingredients Soy Milk Tofu Tempeh Miso Soy SauceHydrolyzed Vegetable Protein Whipping Protein Processed Soy ProteinProducts Full Fat and Defatted Flours Soy Grits Soy Hypocotyls SoybeanMeal Soy Milk Soy Protein Isolates Soy Protein Concentrates Textured SoyProteins Textured Flours and Concentrates Textured Concentrates TexturedIsolates ^(a)See Soy Protein Products: Characteristics, NutritionalAspects and Utilization (1987). Soy Protein Council.

“Processing” refers to any physical and chemical methods used to obtainthe products listed in Table 1A and includes, but is not limited to,heat conditioning, flaking and grinding, extrusion, solvent extraction,or aqueous soaking and extraction of whole or partial seeds.Furthermore, “processing” includes the methods used to concentrate andisolate soy protein from whole or partial seeds, as well as the varioustraditional Oriental methods in preparing fermented soy food products.Trading Standards and Specifications have been established for many ofthese products (see National Oilseed Processors Association Yearbook andTrading Rules 1991-1992). Products referred to as being “high protein”or “low protein” are those as described by these StandardSpecifications. “NSI” refers to the Nitrogen Solubility Index as definedby the American Oil Chemists' Society Method Ac4 41. “KOH NitrogenSolubility” is an indicator of soybean meal quality and refers to theamount of nitrogen soluble in 0.036 M KOH under the conditions asdescribed by Araba and Dale [(1990) Poult. Sci. 69:76-83]. “White”flakes refer to flaked, dehulled cotyledons that have been defatted andtreated with controlled moist heat to have an NSI of about 85 to 90.This term can also refer to a flour with a similar NSI that has beenground to pass through a No. 100 U.S. Standard Screen size. “Cooked”refers to a soy protein product, typically a flour, with an NSI of about20 to 60. “Toasted” refers to a soy protein product, typically a flour,with an NSI below 20. “Grits” refer to defatted, dehulled cotyledonshaving a U.S. Standard screen size of between No. 10 and 80. “SoyProtein Concentrates” refer to those products produced from dehulled,defatted soybeans by three basic processes: acid leaching (at about pH4.5), extraction with alcohol (about 55-80%), and denaturing the proteinwith moist heat prior to extraction with water. Conditions typicallyused to prepare soy protein concentrates have been described by Pass[(1975) U.S. Pat. No. 3,897,574; Campbell et al., (1985) in New ProteinFoods, ed. by Altschul and Wilcke, Academic Press, Vol. 5, Chapter 10,Seed Storage Proteins, pp 302-338]. “Extrusion” refers to processeswhereby material (grits, flour or concentrate) is passed through ajacketed auger using high pressures and temperatures as a means ofaltering the texture of the material. “Texturing” and “structuring”refer to extrusion processes used to modify the physical characteristicsof the material. The characteristics of these processes, includingthermoplastic extrusion, have been described previously [Atkinson (1970)U.S. Pat. No. 3,488,770, Horan (1985) In New Protein Foods, ed. byAltschul and Wilcke, Academic Press, Vol. 1A, Chapter 8, pp 367-414].Moreover, conditions used during extrusion processing of complexfoodstuff mixtures that include soy protein products have been describedpreviously [Rokey (1983) Feed Manufacturing Technology III, 222-237;McCulloch, U.S. Pat. No. 4,454,804].

TABLE 1B Generalized Steps for Soybean Oil and Byproduct ProductionProcess Impurities Removed and/or Step Process By-Products Obtained # 1soybean seed # 2 oil extraction meal # 3 Degumming lecithin # 4 alkalior physical refining gums, free fatty acids, pigments # 5 water washingsoap # 6 Bleaching color, soap, metal # 7 (hydrogenation) # 8(winterization) stearine # 9 Deodorization free fatty acids,tocopherols, sterols, volatiles # 10  oil products

More specifically, soybean seeds are cleaned, tempered, dehulled, andflaked, thereby increasing the efficiency of oil extraction. Oilextraction is usually accomplished by solvent (e.g., hexane) extractionbut can also be achieved by a combination of physical pressure and/orsolvent extraction. The resulting oil is called crude oil. The crude oilmay be degummed by hydrating phospholipids and other polar and neutrallipid complexes that facilitate their separation from the nonhydrating,triglyceride fraction (soybean oil). The resulting lecithin gums may befurther processed to make commercially important lecithin products usedin a variety of food and industrial products as emulsification andrelease (i.e., antisticking) agents. Degummed oil may be further refinedfor the removal of impurities (primarily free fatty acids, pigments andresidual gums). Refining is accomplished by the addition of a causticagent that reacts with free fatty acid to form soap and hydratesphosphatides and proteins in the crude oil. Water is used to wash outtraces of soap formed during refining. The soapstock byproduct may beused directly in animal feeds or acidulated to recover the free fattyacids. Color is removed through adsorption with a bleaching earth thatremoves most of the chlorophyll and carotenoid compounds. The refinedoil can be hydrogenated, thereby resulting in fats with various meltingproperties and textures. Winterization (fractionation) may be used toremove stearine from the hydrogenated oil through crystallization undercarefully controlled cooling conditions. Deodorization (principally viasteam distillation under vacuum) is the last step and is designed toremove compounds which impart odor or flavor to the oil. Other valuablebyproducts such as tocopherols and sterols may be removed during thedeodorization process. Deodorized distillate containing these byproductsmay be sold for production of natural vitamin E and other high-valuepharmaceutical products. Refined, bleached, (hydrogenated, fractionated)and deodorized oils and fats may be packaged and sold directly orfurther processed into more specialized products. A more detailedreference to soybean seed processing, soybean oil production, andbyproduct utilization can be found in Erickson, Practical Handbook ofSoybean Processing and Utilization, The American Oil Chemists' Societyand United Soybean Board (1995). Soybean oil is liquid at roomtemperature because it is relatively low in saturated fatty acids whencompared with oils such as coconut, palm, palm kernel, and cocoa butter.

Plant and microbial oils containing PUFAs that have been refined and/orpurified can be hydrogenated, thereby resulting in fats with variousmelting properties and textures. Many processed fats (including spreads,confectionary fats, hard butters, margarines, baking shortenings, etc.)require varying degrees of solidity at room temperature and can only beproduced through alteration of the source oil's physical properties.This is most commonly achieved through catalytic hydrogenation.

Hydrogenation is a chemical reaction in which hydrogen is added to theunsaturated fatty acid double bonds with the aid of a catalyst such asnickel. For example, high oleic soybean oil contains unsaturated oleic,linoleic, and linolenic fatty acids, and each of these can behydrogenated. Hydrogenation has two primary effects. First, theoxidative stability of the oil is increased as a result of the reductionof the unsaturated fatty acid content. Second, the physical propertiesof the oil are changed because the fatty acid modifications increase themelting point resulting in a semi-liquid or solid fat at roomtemperature.

There are many variables which affect the hydrogenation reaction, whichin turn alter the composition of the final product. Operating conditionsincluding pressure, temperature, catalyst type and concentration,agitation, and reactor design are among the more important parametersthat can be controlled. Selective hydrogenation conditions can be usedto hydrogenate the more unsaturated fatty acids in preference to theless unsaturated ones. Very light or brush hydrogenation is oftenemployed to increase stability of liquid oils. Further hydrogenationconverts a liquid oil to a physically solid fat. The degree ofhydrogenation depends on the desired performance and meltingcharacteristics designed for the particular end product. Liquidshortenings (used in the manufacture of baking products, solid fats andshortenings used for commercial frying and roasting operations) and basestocks for margarine manufacture are among the myriad of possible oiland fat products achieved through hydrogenation. A more detaileddescription of hydrogenation and hydrogenated products can be found inPatterson, H. B. W., Hydrogenation of Fats and Oils: Theory andPractice. The American Oil Chemists' Society (1994).

Hydrogenated oils have become somewhat controversial due to the presenceof trans-fatty acid isomers that result from the hydrogenation process.Ingestion of large amounts of trans-isomers has been linked withdetrimental health effects including increased ratios of low density tohigh density lipoproteins in the blood plasma and increased risk ofcoronary heart disease.

In a further aspect, the present invention concerns an isolatedpolynucleotide comprising:

(a) a nucleotide sequence encoding a polypeptide having diacylglycerolacyltransferase activity wherein the polypeptide has at least 80% aminoacid identity, based on the Clustal V method of alignment, when comparedto an amino acid sequence as set forth in SEQ ID NOs: 135, 136, 147,162, 176, 215, 234, 255, 265, 272, 299, 304, 306, 308, 310, 312, 314,316, 318, or 320;

(b) a nucleotide sequence encoding a polypeptide having diacylglycerolacyltransferase activity, wherein the nucleotide sequence has at least80% sequence identity, based on the BLASTN method of alignment, whencompared to a nucleotide sequence as set forth in SEQ ID NO: 133, 134,146, 161, 175, 214, 233, 264, 271, 298, 303, 305, 307, 309, 311, 313,315, 317, 319, 350, or 362;

(c) a nucleotide sequence encoding a polypeptide having diacylglycerolacyltransferase activity, wherein the nucleotide sequence hybridizesunder stringent conditions to a nucleotide sequence as set forth in SEQID NO: 133, 134, 146, 161, 175, 214, 233, 264, 271, 298, 303, 305, 307,309, 311, 313, 315, 317, 319, 350, or 362; or

(d) a complement of the nucleotide sequence of (a), (b) or (c), whereinthe complement and the nucleotide sequence consist of the same number ofnucleotides and are 100% complementary.

It a related aspect, the present invention concerns an isolatedpolynucleotide comprising:

(a) a nucleotide sequence encoding a polypeptide having diacylglycerolacyltransferase activity wherein the polypeptide is set forth in SEQ IDNOs:135, 136, 147, 162, 176, 215, 234, 255, 265, 272, 299, 304, 306,308, 310, 312, 314, 316, 318, 320, 322, 351, or 363;

(b) a nucleotide sequence encoding a polypeptide having diacylglycerolacyltransferase activity, wherein the nucleotide sequence is set forthin SEQ ID NO: 133, 134, 146, 161, 175, 214, 233, 264, 271, 298, 303,305, 307, 309, 311, 313, 315, 317, 319, 319, 350, or 362; or

(c) a complement of the nucleotide sequence of (a) or (b) wherein thecomplement and the nucleotide sequence consist of the same number ofnucleotides and are 100% complementary.

Further, the aforementioned isolated polynucleotide encoding a DGAT canbe obtained from one or more oleaginous organism. These oleaginousorganisms can be, but are not limited to, Torulaspora delbrueckii,Pichia anomala, Debaryomyces hansenii, Candida zeylanoides, Lipomycesstarkeyi, Mucor circinelloides, Phaffia rhodozyma, Rhodotorula glutinis,Cryptococcus curvatus, and Mortierella alpina.

Recombinant DNA construct comprising the isolated nucleic acid fragmentencoding diacylglycerol acyltransferase can be operably linked to atleast one regulatory sequence, and can be incorporated into a cell. Thecell may be from an oilseed plant.

In a related aspect the present invention concerns a method forincreasing the total fatty acid content of an oilseed comprising:

(a) transforming at least one oilseed cell with the above mentionedrecombinant construct;

(b) selecting the transformed oilseed cell(s) of step (a) having anincreased total fatty acid content when compared to the total fatty acidcontent of a non-transgenic, null segregant oilseed.

In a final aspect the present invention concerns a fungal cell, oroleaginous microbial organism, comprising a recombinant DNA constructcomprising any isolated nucleic acid fragment encoding anydiacylglycerol acyltransferase of the present invention. Further, thefungal cell can be, but is not limited to, Yarrowia, Candida,Rhodotorula, Rhodosporidium, Cryptococcus, Trichosporon and Lipomyces.

EXAMPLES

The present invention is further defined in the following Examples, inwhich parts and percentages are by weight and degrees are Celsius,unless otherwise stated. It should be understood that these Examples,while indicating preferred embodiments of the invention, are given byway of illustration only. From the above discussion and these Examples,one skilled in the art can ascertain the essential characteristics ofthis invention, and without departing from the spirit and scope thereof,can make various changes and modifications of the invention to adapt itto various usages and conditions. Thus, various modifications of theinvention in addition to those shown and described herein will beapparent to those skilled in the art from the foregoing description.Such modifications are also intended to fall within the scope of theappended claims.

The meaning of abbreviations is as follows: “sec” means second(s), “min”means minute(s), “h” means hour(s), “d” means day(s), “μL” meansmicroliter(s), “mL” means milliliter(s), “L” means liter(s), “μM” meansmicromolar, “mM” means millimolar, “M” means molar, “mmol” meansmillimole(s), “μmole” mean micromole(s), “g” means gram(s), “μg” meansmicrogram(s), “ng” means nanogram(s), “U” means unit(s), “bp” means basepair(s) and “kB” means kilobase(s).

Example 1 Expression of Yarrowia lipolytica DGAT Genes in Saccharomycescerevisiae

The DGAT1 gene (SEQ ID NO:1) of Yarrowia lipolytica was excised fromplasmid vector pYDA1 (SEQ ID NO:2) by restriction digestion with NcoIand NotI. The ends of DNA fragment were completely filled in using T4DNA polymerase (Promega, Madison, Wis., USA) and ligated into the uniqueNot I site of pY75 (SEQ ID NO:3). Prior to its use for cloning the pY75vector had been linearized with NotI, filled in with T4 DNA polymeraseand dephosphorylated with shrimp alkaline phosphatase (NEB, Beverly,Mass., USA). Plasmid DNA was isolated using standard techniques andrestriction digests with EcoRI were conducted to identify plasmid clonesin which the start codon was in proximity to the 3′ end of the GPDpromoter in pY75 (sense orientation of the DGAT1 gene). This plasmid ishenceforth referred to as pY75 YL DGAT1 (SEQ ID NO:4). The constructionof pYDA1 is described in PCT Publication No. WO 2006/052914, which ishereby incorporated as reference.

The yeast episomal plasmid (YEp)-type vector pRS425 (SEQ ID NO:5)(Christianson et al., Gene 110:119-122 (1992)) contains sequences fromthe Saccharomyces cerevisiae 2 micron endogenous plasmid, a LEU2selectable marker and sequences based on the backbone of amultifunctional phagemid, pBluescript II SK(+). The Saccharomycescerevisiae strong, constitutive glyceraldehyde-3-phosphate dehydrogenase(GPD) promoter was cloned between the SacII and SpeI sites of pRS425 inthe same way as described by Jia et al. (Physiol. Genomics 3:83-92(2000)) to produce pGPD-425 (SEQ ID NO:6). A Nod site was introducedinto the BamHI site of pGPD-425, thus giving a NotI site flanked byBamHI sites, and this plasmid was called pY75 (SEQ ID NO:3)

The DGAT2 gene was PCR amplified from the genome of Yarrowia lipolytica(ATCC Accession No. 20362) as follows. Yeast cells were grown on solidYPD medium for 72 h. Cells were resuspended in 200 μL of DNA extractionbuffer (100 mM Tris pH 7.5, 10 mM EDTA, 100 mM NaCl, 0.1% Triton X-100)and supplemented with 2-5 glass beads (3 mm diameter) and approximately0.1 g of glass beads (0.5 mm diameter). The yeast cell suspension wasmixed vigorously using a vortex mixer and incubated at 75° C. for 25min. The lysate was cooled to room temperature and cleared bycentrifugation.

The following two oligonucleotide primers were used to generate a PCRfragment of approximately 1600 bp:

oYLDGAT2-1: (SEQ ID NO: 7) GCGGCCGCATGACTATCGACTCACAATACTACAAGT, andoYLDGAT2-2: (SEQ ID NO: 8) GCGGCCGCTTACTCAATCATTCGGAACTCTGGGGCT.Briefly, a PCR reaction mixture (100 μL) containing 2.5 mM MgCl₂, 2 mMdNTPs, 10 mM Tris/HCl (pH 8.8), 50 mM KCl, 0.08% Nonidet P40, 1 μM ofoYLDGAT2-1 (SEQ ID NO:7) and oYLDGAT2-2 (SEQ ID NO:8), 10 U Taqpolymerase (Fermentas, Hanover, Md.), and 2 μL of yeast lysate wascreated. The PCR mixture was divided into four 25 μL aliquots andamplification was carried out for 35 cycles, each comprising 45 sec at94° C., 45 sec at the respective annealing temperature, and 1 min at 72°C. PCR products were gel-purified and cloned into pGEM T-easy (Promega)using manufacturer instructions.

Ten independent plasmid clones were completely sequenced. The consensussequence of this analysis is set forth as SEQ ID NO:9. This DNA sequencediffers from the DGAT2 sequence disclosed in PCT Publication No. WO2005/003322 at two different nucleotide positions. The difference in DNAsequence affects nt 448 and nt 672 of the DGAT2 open reading frame. Theformer nt difference changes the predicted amino acid sequence of theDGAT protein. It replaces a serine found in the DGAT sequence disclosedin PCT Publication No. WO 2005/003322 with a threonine residue. Thesecond sequence difference does not change the amino acid sequence fromthe one disclosed in PCT Publication No. WO 2005/003322. The differencebetween the Yarrowia lipolytica DGAT2 sequence disclosed herein and thatof PCT Publication No. WO 2005/003322 can be attributed to the differentYarrowia lipolytica strains that were used for DGAT2 gene isolation. Thepredicted amino acid sequence of the DGAT protein of strain ATCCAccession No. 20362 is set forth as SEQ ID NO:10.

The DGAT gene (SEQ ID NO:9) was excised as a Not I restriction fragmentfrom the pGEM T-easy vector and ligated to NotI linearized,dephosphorylated DNA of pY75 (SEQ ID NO:3). Plasmid DNA was isolatedform recombinant clones and restriction digestion with Sad and Padallowed to identify clones in which the start codon of the DGAT2 genewas in proximity to the 3′ end of the GPD promoter in pY75 (senseorientation of the DGAT2 gene). This plasmid is henceforth referred toas pY75 YL DGAT2 (SEQ ID NO:11).

Plasmid DNA of pY75 YL DGAT1 (SEQ ID NO:4) and the empty pY75 vectorwere transformed into the Saccharomyces cerevisiae stain INVSC1(Invitrogen, USA) using standard methods (Gietz, R. Daniel; Woods, RobinA., Meth. Enzymol. 350:87-96 (2002)). Recombinant yeast colonies wereselected on DOBA media supplemented with CSM-leu (Qbiogene, Carlsbad,Calif.). Five 50 mL cultures of DOBA media supplemented with CSM-leuwere inoculated with five independently generated colonies and grown and30° C. for 72 h. Cells were harvested by centrifugation and resuspendedin medium identical to the DOBA medium described above with theexception that ammonium sulfate as nitrogen source was omitted. Cultureswere grown for additional 60 h, cells were harvested by centrifugation.Cells were frozen on dry ice and lyophilized.

Total fatty acid content of each yeast cell sample was measured intriplicates as follows. Approximately 5-15 mg of yeast powder wereweighed into the bottom of a 13×100 mm glass culture tube with screw capand Teflon seal. 5 μL of a stock solution of 17:0 TAG (10 mg/mL intoluene) was added followed by addition of 500 μL 5% sulfuric acid inmethanol (anhydrous). Samples were incubated at 95° C. for 1.5 h.Subsequently, tubes were allowed to cool to room temperature after which1 mL of 1 M sodium chloride was added followed by mixing. 1 mL ofheptane was added, contents were mixed and samples were spun briefly tomediate phase separation. Approximately 500 μL of the organic phase wastransferred to a GC vial. Fatty acid methyl esters were analyzed by gaschromatography. 4 μL of heptane extract were analyzed on Hewlett-Packard6890 Gas Chromatograph fitted with an Omegawax 320 fused silicacapillary column (Supelco Inc., Catalog No. 24152). The oven temperaturewas programmed to hold at 220° C. for 2.7 min, increase to 240° C. at 20C/min and then hold for an additional 2.3 min. Carrier gas was suppliedby a Whatman hydrogen generator. Retention times were compared to thosefor methyl esters of standards commercially available (Nu-Chek Prep,Inc. catalog #U-99-A).

Plasmid DNA of pY75 YL DGAT2 (SEQ ID NO:11) and the empty pY75 vectorwere transformed into the Saccharomyces cerevisiae strain INVSC1 andtotal fatty acid content of recombinant yeast cultures was analyzed asdescribed previously. The findings related to over expression of bothDGAT genes in yeast are summarized in TABLE 2.

TABLE 2 Total Fatty Acid Content of Saccharomyces cerevisea Culturesaverage stdv % palmitic % palmitoleic % stearic % oleic FAME FAME FAMEacid acid acid acid (% DCW) stdv (% DCW) (% DCW) pY75 YL 19.1 38.4 7.534.9 12.8 2.2 DGAT1 19.4 38.8 7.4 34.3 12.3 0.4 19.2 38.4 7.6 34.8 12.20.4 19.3 38.6 7.5 34.6 11.5 0.1 19.1 38.3 7.7 34.9 10.9 0.6 11.9 0.8pY75 17.9 37.8 7.9 36.4 10.7 1.0 18.2 38.2 7.8 35.8 9.7 0.6 17.9 41.06.8 34.2 8.7 0.3 17.2 40.5 6.9 35.4 8.7 0.6 18.1 41.1 6.9 33.9 8.5 0.29.3 0.9 pY75 YL 31.8 34.0 14.2 20.0 17.1 0.2 DGAT2 31.4 33.1 14.8 20.615.9 0.6 30.7 32.8 14.7 21.8 13.6 1.1 28.9 34.2 13.5 23.5 12.4 1.4 29.234.1 13.5 23.1 11.8 1.7 14.2 2.3 pY75 19.7 37.0 9.6 33.7 7.0 0.4 20.036.4 9.8 33.7 6.8 0.0 19.6 37.0 9.6 33.8 6.6 0.6 19.7 37.1 9.5 33.6 6.40.2 19.3 36.9 9.7 34.1 6.4 0.3 6.6 0.2

TABLE 2 shows that there is a significant increase of total fatty acidsin yeast cells harboring the pY75 YL DGAT1 (SEQ ID NO:4) compared tocells that only contain the empty pY75 plasmid. The average fatty aciddry cell weigt (DCW) percentage of five independent cultures is 11.9%compared to 9.3% for vector controls grown under identical conditions.In summary, there is a 28% increase in total fatty acid production.Moreover, there is a slight alteration in the fatty acid profileassociated with YL DGAT 1 expression characterized by an increase inpalmitic acid.

Constitutive expression of YL DGAT2 under nitrogen starvation increasedtotal fatty acid content by 110% compared to a vector control grownunder identical conditions. Total fatty acid content of the vector onlycontrol was 6.6% whereas the average fatty acid content of the YL DGAT2transformants was 14.2%. The fatty acid profile changed as result of YLDGAT2 expression. Palmitic acid content increased significantlyaccompanied by a moderate decrease in palmitoleic. In addition, stearicacid content increased significantly accompanied by a clear decrease inoleic content. Taken together the results show that YL DGATsover-expression in yeast under conditions of increased carbon/nitrogenratios lead to increased fatty acid accumulation. The overexpressed YLDGAT enzymes are able to augment endogenous DGAT activity inSaccharomyces cerevisiae. Similar experiments were repeated with bothDGAT genes two more times. A difference in fatty acid content between YLDGAT culture and vector control could be observed every time and was atleast 8% and 14.3% for YL DGAT1 (SEQ ID NO:1) and YL DGAT2 (SEQ IDNO:9), respectively.

Example 2 Cloning the Yarrowia lipolytica DGAT1 And DGAT2 into Yarrowialipolytica Expression Vectors

The present Example describes the generation of pFBAIN-YDG1 andpFBAIN-YDG2, comprising a chimeric FBAINm::YDGAT1::PEX20 gene and achimeric FBAINm::YDGAT2::PEX20 gene, respectively (FIGS. 1A and 1B).These were designed for overexpression of the DGAT1 and DGAT2 inYarrowia lipolytica.

Oligonucleotides YDGAT1-F (SEQ ID NO:12) and YDGAT-R (SEQ ID NO:13) weredesigned and synthesized to allow amplification of the DGAT1 ORF fromYarrowia lipolytica genomic DNA (isolated from strain ATCC Accession No.20362, purchased from the American Type Culture Collection (Rockville,Md.)), while oligonucleotides YDGAT2-F (SEQ ID NO:14) and YDGAT2-R (SEQID NO:15) were designed and synthesized to allow the amplification ofthe DGAT2 ORF.

The PCR reactions, with Yarrowia lipolytica genomic DNA as template,were individually carried out in a 50 μL total volume comprising: 1 μLeach of 20 μM forward and reverse primers, 1 μL genomic DNA (100 ng), 10μL 5×PCR buffer, 1 μL dNTP mix (10 μM each), 35 μL water and 1 μLPhusion polymerase (New England Biolabs, Inc., Ipswich, Mass.).Amplification was carried out at 98° C. for 1 min, followed by 30 cyclesat 98° C. for 10 sec, 55° C. for 10 sec, and 72° C. for 30 sec, followedby a final elongation cycle at 72° C. for 5 min. A 1603 bp DNA fragment(SEQ ID NO:16) and a 1567 bp fragment (SEQ ID NO:17) were generated thatcontained the DGAT1 and DGAT2 ORFs, respectively.

The PCR fragments were purified with Qiagen PCR purification kitsfollowing the manufacturer's protocol. Purified DNA samples weredigested with NcoI and NotI, purified with a Qiagen reaction clean-upkit, and then directionally ligated with NcoI/NotI digested pFBAIN-MOD-1(FIG. 1C; SEQ ID NO:18). Specifically, the ligation reaction contained:10 μL 2× ligation buffer, 1 μL T4 DNA ligase (Promega), 4 μL (˜300 ng)of either the 1600 bp fragment (i.e., DGAT1; SEQ ID NO:16) or the 1564bp fragment (i.e., DGAT2; SEQ ID NO:17) and 1 μL pFBAIN-MOD-1 (˜150 ng).The reaction mixtures were incubated at room temperature for 2 h andused to transform E. coli Top10 competent cells (Invitrogen). PlasmidDNA from transformants was recovered using a Qiagen Miniprep kit.Correct clones were identified by restriction mapping and the finalconstructs were designated “pFBAIN-YDG1” and “pFBAIN-YDG2”,respectively.

Thus, pFBAIN-YDG1 (FIG. 1A; SEQ ID NO:19) thereby contained thefollowing components:

TABLE 3 Components of Plasmid pFBAIN-YDG1 (SEQ ID NO: 19) RE Sites AndNucleotides Within SEQ ID Description of Fragment and NO: 19 ChimericGene Components BglII-BsiWI FBAINm::YDG1::PEX20, comprising: (6040-301) FBAINm: Yarrowia lipolytica FBAINm promoter (PCT Publication No. WO2005/049805) YDG1: Y. lipolytica DGAT1 ORF (SEQ ID NO: 16) Pex20: Pex20terminator sequence of Yarrowia Pex20 gene (GenBank Accession No.AF054613) PacI-BglII Yarrowia URA3 (GenBank Accession No. AJ306421)(4533-6040) (3123-4487) Yarrowia autonomous replicating sequence 18(ARS18; GenBank Accession No. M91600 and No. A17608) (2464-2864) f1origin (1424-2284) ampicillin-resistance gene (Amp^(R)) for selection inE. coli  (474-1354) ColE1 plasmid origin of replicationPlasmid pFBAIN-YDG2 (FIG. 1B; SEQ ID NO:20) contained componentsidentical to those of pFBAIN-YDG1, with the exception that the Yarrowialipolytica DGAT2 ORF (SEQ ID NO:17; identified as YDG2 on FIG. 1B) waspresent instead of the Yarrowia lipolytica DGAT1 ORF in pFBAIN-YDG1.

The term “FBAINm promoter” or “FBAINm promoter region” is a modifiedversion of the FBAIN promoter (infra), wherein FBAINm has a 52 bpdeletion between the ATG translation initiation codon and the intron ofthe FBAIN promoter (thereby including only 22 amino acids of theN-terminus) and a new translation consensus motif after the intron.Furthermore, while the FBAIN promoter generates a fusion protein whenfused with the coding region of a gene to be expressed, the FBAINmpromoter does not generate such a fusion protein. The FBAIN promoterrefers to the 5′ upstream untranslated region in front of the ‘ATG’translation initiation codon of the Yarrowia lipolyticafructose-bisphosphate aldolase enzyme (E.C. 4.1.2.13) encoded by thefba1 gene and that is necessary for expression, plus a portion of 5′coding region that has an intron of the fba1 gene. These promoters aredescribed in detail in PCT Publication No. WO 2005/049805 and U.S. Pat.No. 7,202,356, which are hereby incorporated by reference in theirentirety.

Example 3 Overexpression of Yarrowia lipolytica DGAT1 And DGAT2 Genes inYarrowia lipolytica Strain Y2224

The present Example describes increased fatty acid content, andmodification to the relative abundance of each fatty acid species, inYarrowia lipolytica strain Y2224 that was transformed to co-expresseither the Yarrowia lipolytica DGAT1 (SEQ ID NO:16) or the Yarrowialipolytica DGAT2 (SEQ ID NO:17). Strain Y2224 is a FOA resistant mutantfrom an autonomous mutation of the Ura3 gene of wild type Yarrowiastrain ATCC Accession No. 20362.

Generation Of Strain Y2224: Strain Y2224 was isolated in the followingmanner: Yarrowia lipolytica ATCC Accession No. 20362 cells from a YPDagar plate (1% yeast extract, 2% bactopeptone, 2% glucose, 2% agar) werestreaked onto a minimal media plate (75 mg/L each of uracil and uridine,6.7 g/L YNB with ammonia sulfate, without amino acid, and 20 g/Lglucose) containing 250 mg/L 5-FOA (5-fluorouracil-6-carboxylic acidmonohydrate; Zymo Research). Plates were incubated at 28° C. and four ofthe resulting colonies were patched separately onto minimal media (MM)plates containing 200 mg/mL 5-FOA and MM plates lacking uracil anduridine to confirm uracil Ura3 auxotrophy.

Transformation of Strain Y2224:

A clone of pFBAIn-YDG1, a clone of pFBAIn-YDG2 and control plasmidpFBAIN-MOD-1 were transformed into Yarrowia lipolytica strain Y2224 asdescribed below.

Transformation of Yarrowia lipolytica was performed according to themethod of Chen, D. C. et al. (Appl. Microbiol. Biotechnol. 48(2):232-235(1997)), unless otherwise noted. Briefly, Yarrowia was streaked onto aYPD agar plate and grown at 30° C. for approximately 18 h. Several largeloopfuls of cells were scraped from the plate and resuspended in 1 mL oftransformation buffer containing: 2.25 mL of 50% PEG, average MW 3350;0.125 mL of 2 M lithium acetate, pH 6.0; 0.125 mL of 2 M DTT; and 50 μgsheared salmon sperm DNA. Then, approximately 500 ng of linearizedplasmid DNA was incubated in 100 μL of resuspended cells, and maintainedat 39° C. for 1 h with vortex mixing at 15 min intervals.

The cells from each transformation were plated onto minimal media (MM)plates lacking uracil (0.17% yeast nitrogen base (DIFCO Laboratories,Detroit, Mich.) without ammonium sulfate or amino acids, 2% glucose,0.1% proline, pH 6.1, 20 g/L agar) and maintained at 30° C. for 2 days.Three transformants from each transformation plate were used toinoculate individual 25 mL culture in MM medium (0.17% yeast nitrogenbase (DIFCO Laboratories) without ammonium sulfate or amino acids, 2%glucose, 0.1% proline, pH 6.1). Each culture was allowed to grow for 2days at 30° C., then switched into 25 mL of high glucose medium (“HGmedium”, comprising 80 g/L glucose, 27 g/L K₂HPO₄, 6.3 g/L KH₂PO₄, pH˜7.5) and allowed to grow for 5 days at 30° C.

Lipid Analysis:

Total lipids were extracted, and fatty acid methyl esters (FAMEs) wereprepared by trans-esterification, and subsequently analyzed with aHewlett-Packard 6890 GC.

More specifically, for fatty acid analysis, cells were collected bycentrifugation and lipids were extracted as described in Bligh, E. G. &Dyer, W. J. (Can. J. Biochem. Physiol. 37:911-917 (1959)). Fatty acidmethyl esters were prepared by transesterification of the lipid extractwith sodium methoxide (Roughan, G. and Nishida I., Arch Biochem Biophys.276(1):38-46 (1990)) and subsequently analyzed with a Hewlett-Packard6890 GC fitted with a 30-m×0.25 mm (i.d.) HP-INNOWAX (Hewlett-Packard)column. The oven temperature was from 170° C. (25 min hold) to 185° C.at 3.5° C./min.

For direct base transesterification, Yarrowia culture (3 mL) washarvested, washed once in distilled water, and dried under vacuum in aSpeed-Vac for 5-10 min. Sodium methoxide (100 μL of 1%) was added to thesample, and then the sample was vortexed and rocked for 20 min. Afteradding 3 drops of 1 M NaCl and 400 μL hexane, the sample was vortexedand spun. The upper layer was removed and analyzed by GC as describedabove.

Based on the above analyses, lipid content and composition wasdetermined in transformant strains of Y2224, comprising pFBAIn-YDG1,pFBAIn-YDG2 and pFBAIN-MOD-1 (control), respectively, as shown below inTABLE 4. Three independent transformants of each strain were analyzed,while the average results are shown in the rows highlighted in grey.Fatty acids are identified as 16:0 (palmitate), 16:1 (palmitoleic acid),18:0, 18:1 (oleic acid) and 18:2 (LA); and the composition of each ispresented as a % of the total fatty acids.

“% FAME/DCW” represents the percent fatty acid methyl ester/dry cellweight. Dry cell weight was determined by collecting cells from 10 mL ofculture via centrifugation, washing the cells with water once to removeresidue medium, drying the cells in a vacuum oven at 80° C. overnight,and weighing the dried cells. The total amount of fatty acid methylesters in a sample was determined by comparing the areas of all peaks inthe GC profile with the peak area of an added known amount of internalstandard C15:0 fatty acid.

TABLE 4 Lipid Content A\and Composition in Yarrowia Strain Y2224Overexpressing YDGAT1 And YDGAT2

GC analyses showed that there was a significant increase of total fattyacids in cells carrying pFBAIn-YDG1, as compared to cells carryingpFBAIn-MOD-1. The average fatty acid increased from 21.39% FAME/DCW inthe control to 23.70% FAME/DCW in cells expressing YDGAT1 (i.e., a 10.8%increase). Furthermore, there was also an increase in the amount ofC16:1 and C18:1 fatty acids and a decrease of C18:2 fatty acid (TABLE4).

Cells carrying pFBAIn-YDG2 also had a large increase in total fatty acidcontent relative to the control, resulting in an average of 27.32%FAME/DCW (representing an increase of 27.7%). The distribution of fattyacid species also changed significantly. Specifically, the amount ofC18:0 and C18:1 was increased, whereas the amount of C16:0, C16:1 andC18:2 decreased.

Collectively, these results demonstrate that overexpression of theYarrowia lipolytica DGAT1 or DGAT2 impacts both total lipid content andthe relative abundance of each fatty acid species.

Example 4 Expression of Yarrowia lipolytica DGAT Genes in ArabidopsisSeed

A binary vector suitable for agrobacterium-mediated transformation wasgenerated as follows. Various restriction sites were added, through anumber of cloning steps, to the ends of the Bcon/NotI/Phas3′ cassettefrom KS123 (SEQ ID NO:21), which was previously described in PCTPublication No. WO 02/008269 (the contents of which are herebyincorporated by reference). Briefly, a DNA fragment (cal a24-4; SEQ IDNO:22) was amplified from plasmid CalFad2-2 (described in PCTPublication No. WO 01/12800) using primers oCal-15 (SEQ ID NO:23) andoCal-6 (SEQ ID NO:24). DNA fragment cal a24-4 (SEQ ID NO:22) wasdigested with BglII and BamHI and cloned into the BamHI site of pKS123to give pKR53B (SEQ ID NO:25). The XbaI/SbfI fragment of pKR53B,containing the Bcon/NotI/Phas3′ cassette was cloned into the XbaI/SbfIfragment of pKR72 (ATCC Accession No. PTA-6019; SEQ ID NO:26) containingthe bacterial hygromycin phosphotransferase gene, to give pKR85 (SEQ IDNO:27). The features of pKR72 are as follows. A starting plasmid pKR72(ATCC Accession No. PTA-6019; SEQ ID NO:26), a derivative of pKS123which was previously described in PCT Publication No. WO 02/008269 (thecontents of which are hereby incorporated by reference), contains thehygromycin B phosphotransferase gene (HPT) (Gritz, L. and Davies, J.,Gene 25:179-188 (1983)), flanked by the T7 promoter and transcriptionterminator (T7prom/hpt/T7term cassette), and a bacterial origin ofreplication (ori) for selection and replication in bacteria (e.g., E.coli). In addition, pKR72 also contains the hygromycin Bphosphotransferase gene, flanked by the 35S promoter (Odell et al.,Nature 313:810-812 (1985)) and NOS 3′ transcription terminator (Depickeret al., J. Mol. Appl. Genet. 1:561-570 (1982)) (35S/hpt/NOS3′ cassette)for selection in plants such as soybean. pKR72 also contains a NotIrestriction site, flanked by the promoter for the α′ subunit ofβ-conglycinin (Beachy et al., EMBO J. 4:3047-3053 (1985)) and the 3′transcription termination region of the phaseolin gene (Doyle et al., J.Biol. Chem. 261:9228-9238 (1986)), thus allowing for strongtissue-specific expression in the seeds of soybean of genes cloned intothe NotI site.

The Bcon/NotI/Phas3′ cassette was amplified from plasmid pKR85 (SEQ IDNO:27) using primers oKR85-1 (SEQ ID NO:28) and oKR85-2 (SEQ ID NO:29)and the resulting DNA fragment was cloned into PCR-Script® (Stratgene)following the manufacture's protocol, to give pPCR85 (SEQ ID NO:30).

The EcoRI/Bg/II fragment of pPCR85, containing the Bcon/NotI/Phas3′cassette was cloned into the EcoRI/BamHI fragment of plasmid pZS199 (PCTPublication No. WO 93/11245; also U.S. Pat. No. 5,952,544 which waspublished on Jun. 10, 1993; the disclosures of which are herebyincorporated by reference), containing the Arabidopsis binary vectorbackbone to produce pKR91 (SEQ ID NO:31).

The Bcon/NotI/Phas3′ cassette was removed from pKR91 by digestion withAscI and the re-ligated binary vector containing a unique AscI cloningsite was produced called pKR92 (SEQ ID NO:32).

Construction of pKR92YL DGAT2:

The construction of expression plasmid KS362 is described in Example 5.An expression cassette which harbors the YL DGAT2 gene, fused tobetaconglycinin promoter and the phaseolin terminator and DsRed genefused to Kti promoter and terminator sequences was excised from KS362 asa 6.4 kb Ascl fragment. This DNA was ligated to Ascl linearized,dephosphorylated pKR92 vector DNA to give pKR92YL DGAT2 (SEQ ID NO:33).

Construction of pKR92YL DGAT1/YL DGAT2:

The construction of expression plasmid KS364 is described in Example 5.An expression cassette in which YL DGAT1 (SEQ ID NO:1) and YL DGAT2 (SEQID NO:9) genes are fused to identical sequence of the phaseolinterminator and to glycinin 1 and betaconglycinin promoters respectivelywas excised from KS364 as a 7 kb AscI fragment. This DNA was ligated toAscI linearized, dephosphorylated pKR92 vector DNA to give pKR92YLDGAT1/YL DGAT2 (SEQ ID NO:34)

Generation and Analysis of Transgenic Arabidospis Lines:

Plasmid DNA of pKR92YL DGAT2 and pKR92YL DGAT1/YL DGAT2 was introducedinto Agrobacterium tumefaciens NTL4 (Luo et al, Molecular Plant-MicrobeInteractions 14(1):98-103 (2001)) by electroporation. Briefly, 1 μgplasmid DNA was mixed with 100 μL of electro-competent cells on ice. Thecell suspension was transferred to a 100 μL electro oration curette (1mm gap width) and electro orated using a BIORAD electro orator set to 1kV, 400Ω and 25 μF. Cells were transferred to 1 mL LB medium andincubated for 2 h at 30° C. Cells were plated onto LB medium containing50 μg/mL kanamycin. Plates were incubated at 30° C. for 60 h.Recombinant agrobacterium cultures (500 mL LB, 50 μg/mL kanamycin) wereinoculated from single colonies of transformed agrobacterium cells andgrown at 30° C. for 60 h. Cells were harvested by centrifugation(5000×g, 10 min) and resuspended in 1 L of 5% (W/V) sucrose containing0.05% (V/V) Silwet. Arabidopsis plants were grown in soil at a densityof 30 plants per 100 cm² pot in metromix 360 soil mixture for 4 weeks(22° C., permanent light, 100 μE m⁻²s⁻¹). Plants were repeatedly dippedinto the agrobacterium suspension harboring the binary vectors and keptin a dark, high humidity environment for 24 h. Plants were grown forfour to five weeks under standard plant growth conditions describedabove and plant material was harvested and dried for one week at ambienttemperatures in paper bags. Seeds were harvested using a 0.425 mm meshbrass sieve.

Cleaned Arabidopsis seeds (2 g, corresponding to about 100,000 seeds)were sterilized by washes in 45 mL of 80% ethanol, 0.01% triton X-100,followed by 45 mL of 30% (V/V) household bleach in water, 0.01% tritonX-100 and finally by repeated rinsing in sterile water. Aliquots of20,000 seeds were transferred to square plates (20×20 cm) containing 150mL of sterile plant growth medium comprised of 0.5×MS salts, 1.0% (W/V)sucrose, 0.05 MES/KOH (pH 5.8), 200 μg/mL timentin, and 50 μg/mLkanamycin solidified with 10 g/L agar. Homogeneous dispersion of theseed on the medium was facilitated by mixing the aqueous seed suspensionwith an equal volume of melted plant growth medium. Plates wereincubated under standard growth conditions for ten days.Kanamycin-resistant seedlings were transferred to plant growth mediumwithout selective agent and grown to maturity for 8-10 weeks (22° C.,permanent light dark, 100-200 μE m⁻²s⁻¹). Plants were grown in flatswith 36 inserts. In every flat at least six untransformed wild typecontrol plants were grown next to approximately thirty T2 plants. Seedswere harvested from individual plants and seed oil content was measuredby NMR.

NMR Based Analysis of Seed Oil Content:

Seed oil content was determined using a Maran Ultra NMR analyzer(Resonance Instruments Ltd, Whitney, Oxfordshire, UK). Samples (eitherindividual soybean seed or batches of Arabidopsis seed ranging in weightbetween 5 and 200 mg) were placed into pre-weighed 2 mL polypropylenetubes (Corning Inc, Corning N.Y., USA; Part no. 430917) previouslylabeled with unique bar code identifiers. Samples were then placed into96 place carriers and processed through the following series of steps byan Adept Cobra 600 SCARA robotic system.

-   -   1. pick up tube (the robotic arm was fitted with a vacuum pickup        devise)    -   2. read bar code    -   3. expose tube to antistatic device (ensured that Arabidopsis        seed were not adhering to the tube walls)    -   4. weigh tube (containing the sample), to 0.0001 g precision.    -   5. NMR reading; measured as the intensity of the proton spin        echo 1 msec after a 22.95 MHz signal had been applied to the        sample (data was collected for 32 NMR scans per sample)    -   6. return tube to rack    -   7. repeat process with next tube        Bar codes, tubes weights and NMR readings were recorded by a        computer connected to the system. Sample weight was determined        by subtracting the polypropylene tube weight from the weight of        the tube containing the sample.

Seed oil content of soybeans seed was calculated as follows:

${\% \mspace{14mu} {oil}\mspace{11mu} \left( {\% \mspace{14mu} {wt}\mspace{14mu} {basis}} \right)} = \frac{\left. {\left( {{NMR}\mspace{14mu} {signal}\text{/}{sample}\mspace{14mu} {wt}\mspace{11mu} (g)} \right) - 70.58} \right)}{351.45}$

Calibration parameters were determined by precisely weighing samples ofsoy oil (ranging from 0.0050 to 0.0700 g at approximately 0.0050 gintervals; weighed to a precision of 0.0001 g) into Corning tubes (seeabove) and subjecting them to NMR analysis. A calibration curve of oilcontent (% seed wt basis; assuming a standard seed weight of 0.1500 g)to NMR value was established.

The relationship between seed oil contents measured by NMR and absoluteoil contents measured by classical analytical chemistry methods wasdetermined as follows. Fifty soybean seed, chosen to have a range of oilcontents, were dried at 40° C. in a forced air oven for 48 h. Individualseeds were subjected to NMR analysis, as described above, and were thenground to a fine powder in a GenoGrinder (SPEX Centriprep (Metuchen,N.J., U.S.A.); 1500 oscillations per minute, for 1 minute). Aliquots ofbetween 70 and 100 mg were weighed (to 0.0001 g precision) into 13×100mm glass tubes fitted with Teflon® lined screw caps; the remainder ofthe powder from each bean was used to determine moisture content, byweight difference after 18 h in a forced air oven at 105° C. Heptane (3mL) was added to the powders in the tubes and after vortex mixingsamples were extracted, on an end-over-end agitator, for 1 h at roomtemperature. The extracts were centrifuged, 1500×g for 10 min, thesupernatant decanted into a clean tube and the pellets were extractedtwo more times (1 h each) with 1 mL heptane. The supernatants from thethree extractions were combined and 50 μL internal standard(triheptadecanoic acid; 10 mg/mL toluene) was added prior to evaporationto dryness at room temperature under a stream of nitrogen gas; standardscontaining 0, 0.0050, 0.0100, 0.0150, 0.0200 and 0.0300 g soybean oil,in 5 mL heptane, were prepared in the same manner. Fats were convertedto fatty acid methyl esters (FAMEs) by adding 1 mL 5% sulfuric acid(v:v. in anhydrous methanol) to the dried pellets and heating them at80° C. for 30 min, with occasional vortex mixing. The samples wereallowed to cool to room temperature and 1 mL 25% aqueous sodium chloridewas added followed by 0.8 mL heptane. After vortex mixing the phaseswere allowed to separate and the upper organic phase was transferred toa sample vial and subjected to GC analysis.

Plotting NMR determined oil contents versus GC determined oil contentsresulted in a linear relationship between 9.66 and 26.27% oil (GCvalues; % seed wt basis) with a slope of 1.0225 and an R² of 0.9744;based on a seed moisture content that averaged 2.6+/−0.8%.

Seed Oil Content of arabidopsis Seed was Calculated as Follows:

mg oil=(NMR signal−2.1112)/37.514%

% oil(% wt basis)=(mg oil/1000)/sample weight)*100

Prior to establishing this formula, Arabidopsis seed oil was extractedas follows. Approximately 5 g of mature Arabidopsis seed (cv Columbia)were ground to a fine powder using a mortar and pestle. The powder wasplaced into a 33×94 mm paper thimble (Ahlstrom #7100-3394; Ahlstrom,Mount Holly Springs, Pa., USA) and the oil extracted duringapproximately 40 extraction cycles with petroleum ether (BP 39.9-51.7°C.) in a Soxhlet apparatus. The extract was allowed to cool and thecrude oil was recovered by removing the solvent under vacuum in a rotaryevaporator. Calibration parameters were determined by precisely weighing11 standard samples of partially purified Arabidopsis oil (samplescontained 3.6, 6.3, 7.9, 9.6, 12.8, 16.3, 20.3, 28.2, 32.1, 39.9 and 60mg of partially purified Arabidopsis oil) weighed to a precision of0.0001 g) into 2 mL polypropylene tubes (Corning Inc, Corning N.Y., USA;Part no. 430917) and subjecting them to NMR analysis. A calibrationcurve of oil content (% seed wt basis) to NMR value was established.Seed for pKR92YL DGAT2 T2 were grown from a total of 293 independentevents alongside 75 wild type controls. Oil content of YL DGAT2transgenics ranged from 27.9-47.3%. Average oil content was 43.7%. Oilcontent of wild type controls ranged from 39.5-49.6%. Average oilcontent of wt controls was 44.8%.

Seed for pKR92YL DGAT1/YL DGAT2 T2 were grown from a total of 295independent events alongside 77 wild type controls. Oil content of YLDGAT1/YL DGAT2 transgenics ranged from 34.5-47.6%. Average oil contentwas 44%. Oil content of wild type controls ranged from 41.3-46.6%.Average oil content of wild type controls was 45%. In summary, thesefindings suggest that seed-specific expression of YL DGAT gene does notincrease oil content of arabidopsis seed.

Analysis of the Fatty Acid Profile of Arabdidopsis Seed Expressing YLDGAT2 Alone or in Combination with YL DGAT1:

GC analysis of FAME was employed to investigate if YL DGAT expressionalters the fatty acid profile of arabidopsis seed. Approximately 100 F2seed were dispensed into individual wells of 96 well strip tubes. Fortransesterification, 50 μL of trimethylsulfonium hydroxide (TMSH) and0.5 mL of hexane were added to the each strip tube and incubated for 30min at room temperature while shaking. Fatty acid methyl esters (1 μLinjected from hexane layer) were separated and quantified using aHewlett-Packard 6890 Gas Chromatograph fitted with an Omegawax 320 fusedsilica capillary column (Catalog #24152, Supelco Inc.). The oventemperature was programmed to hold at 220° C. for 2.6 min, increase to240° C. at 20° C./min and then hold for an additional 2.4 min. Carriergas was supplied by a Whatman hydrogen generator. Retention times werecompared to those for methyl esters of standards commercially available(Nu-Chek Prep, Inc.). Results are summarized in TABLE 5.

TABLE 5 avg % range % oleic oleic pKR92 YL DGAT1/YL DGAT2 18.7 15.2-22.3(n = 28) wild type control 15.2 14.5-15.6 (n = 8) pKR92 YL DGAT2 16.414.9-18.4 (n = 26) wild type control 15.7 15.6-16   (n = 7)Results clearly demonstrate that expression of YL DAGT2 and even more soco-expression of YL DGAT1 and YL DGAT2 in Arabidopsis seed leads toincreased incorporation of oleic acid into seed lipids which providesthe first indication that active YL DGAT1 and YL DGAT2 proteins can beproduced in transgenic seed.

Example 5 Expression of Yarrowia lipo/vtica DGAT Genes in SoybeanSomatic Embryos

TABLE 6 and TABLE 7 list promoter and terminator sequences that wereused in plasmid constructs for seed specific over-expression of YL DGATgenes.

TABLE 6 Seed-specific Promoters Promoter Organism Promoter Referenceβ-conglycinin α′-subunit soybean Beachy et al., EMBO J. 4: 3047-3053(1985) kunitz trypsin inhibitor soybean Jofuku et al., Plant Cell 1:1079-1093 (1989) glycinin Gy1 soybean WO 2004/071467 BD30 (also calledP34) soybean WO 2004/071467

TABLE 7 Transcription Terminators Transcription Terminator OrganismReference phaseolin 3′ bean WO 2004/071467 kunitz trypsin inhibitor 3′soybean WO 2004/071467Construction of a plasmid construct for expression of the YL DGAT1 geneunder control of the glycinin Gy1 promoter (KS349) (FIG. 3).

The isolation of soybean glycinin Gy1 promoter was performed as follows.Based on the sequences of the soybean glycinin Gy1 gene sequence(GenBank Accession No. X15121; SEQ ID NO:35) in the NCBI database, twooligos with either BamHI or NcoI sites at the 5′ ends were designed toamplify the soybean glycinin Gy1 promoter (SEQ ID NO:36). Theoligonucleotide sequences of these two oligos are as follows:

SEQ ID NO: 37 (oGy1-1): CGCGGATCCTAGCCTAAGTACGTACTCAAAATGCCASEQ ID NO: 38 (oGy1-2): GAATTCCCATGGGGTGATGACTGATGAGTGTTTAAGGAC

Plasmid pKS349 was constructed in many steps from a number of differentintermediate vectors. The amplified soybean glycinin Gy1 promoterfragment was digested with BamHI and NcoI, purified and cloned into theBamHI and NcoI sites of p24K-G4G-@SalI (PCT Application No. WO 98/59062)to give pZBL114 (SEQ ID NO:39). The NcoI/KpnI fragment containing GUSwas replaced with an NcoI/KpnI fragment containing a fusion product ofthe soybean GY1 gene (SEQ ID NO:40) and the synthetic barley high lysine8 (BHL8) gene (U.S. Pat. No. 6,800,726 B1) (SEQ ID NO:41;) to makepZBL133 (SEQ ID NO:43). The DNA sequence of the fusion product of soyGY1 gene and BHL8 gene is set forth as SEQ ID NO: 42. The phaseolinterminator was removed from pZBL133 (XbaI/filled in) and replaced withthe phaseolin terminator found in pKS 123 (PCT Application No. WO02/08269) (blunt) to give pKS238 (SEQ ID NO:44). The GY1-BHL8 fusion wasreplaced with native GY1 sequence as follows. pKS238 was digested withKpnI/BglI, the remaining vector band (5.1 kb) was ligated to a DNAfragment (BglI/KpnI) of the native GY1 gene (SEQ ID NO:40) to givepKS240 (SEQ ID NO: 45) The BamHI/SalI fragment containingGy1/GM-GY1/Phas3′ was excised from pKS 240 and ligated to theBamtHI/SalI sites of pKS120 (SEQ ID NO:46) to give pKS242 (SEQ IDNO:47). Plasmid pKS120 is identical to pKS123 (supra) with the exceptionthat the HindIII fragment containing Bcon/NotI/Phas3′ cassette wasremoved. The NcoI/NotI fragment containing GM-GY1 was replaced with theNcoI/NotI fragment containing YL-DGAT1 from pYDA1 to give pKS349 (SEQ IDNO:48).

Construction of a Construct for Expression of the YL DGAT2 Gene Underthe Control of the Betaconglycinin Promoter (KS362) (FIG. 3):

Plasmid pKS362 was constructed in many steps from a number of differentintermediate vectors. The Ascl cassette containing Kti/NotI/Kti3′ frompKS121 (PCT Application No. WO 02/00904) was blunted into the NotI(filled in) site on pBluescript II SK+ (Stratagene) to give pKS121/BS.The NcoI/NotI fragment from pDsRed-Express Vector (Clontech) was bluntedinto the NotI (filled in) site of pKS121/BS to give pDS-RED in KS121/BS(SEQ ID NO:49). The BamHI cassette containing Kti/DsRed/Kti3′ in pDS-REDin KS121/BS (SEQ ID NO:50) was ligated into the BamHI site of pKS123(PCT Application No. WO 02/08269) to give pKS332 (SEQ ID NO:51). Thegene for the YL-DGAT2 was synthesized by PCR with primers to introduceNotI sites at both ends of the gene (see Example 1). The resulting PCRproduct is digested with NotI restriction enzyme and ligated into theNotI site of pKS332 to give pKS362 (SEQ ID NO:52).

Construction of a Control Plasmid (KS352) (FIG. 2):

Based on the sequences of the cloned soybean P34 promoter (WO2004/071467) (SEQ ID NO:53), two oligos with either BamH I or NotI sitesat the 5′ ends were designed to re-amplify the P34 promoter. Theoligonucleotide sequences of these two oligos are shown as follows:

SEQ ID NO:54 (oP34-1): CGCGGATCCAACTAAAAAAAGCTCTCAAATTACATTTTGAGSEQ ID NO:55 (oP34-2): GAATTCGCGGCCGCAACTTGGTGGAAGAATTTTATGATTTGAAAThe re-amplified P34 promoter fragment was digested with BamHI and NotI,purified and cloned into the BamHI and NotI sites of plasmid pZBL115(SEQ ID NO:56) to make pJS89 (SEQ ID NO:57). The pZBL115 plasmidcontains the origin of replication from pRB322, the bacterial HPThygromycin resistance gene driven by T7 promoter and T7 terminator, anda ³⁵S promoter-HPT-Nos3′ gene to serve as a hygromycin resistant plantselection marker. Morteriella alpina delta-6 desaturase gene (U.S. Pat.No. 5,968,809) (SEQ ID NO:58) was cloned into the NotI site of pJS89(SEQID NO:57) in the sense orientation to make the plant expressioncassettes and pJS93 (SEQ ID NO:59).

The P34 promoter was excised from pJS93 (SEQ ID NO:59) using SalI NotIdouble digestion and ligated to SalI/NotI linearized pKS127 vector (U.S.patent application Ser. No. 11/476,510) (SEQ ID NO:60) to give pKS343(SEQ ID NO:61). The BamHI cassette containing Kti/DsRed/Kti3′ in pDS-REDin KS121/BS was blunted and ligated into the HindM (filled in) site ofpKS343 to give pKS352 (SEQ ID NO:62)

Construction of a Plasmid for Co-expression of YL DGAT1 and YL DGAT2(KS364):

Plasmid pKS364 (SEQ ID NO:63) was constructed by ligating the 3.3 kbHindIII cassette containing Bcongl PRO/YL-DGAT2/Phas TER from pKS362(SEQ ID NO:52) into the unique HindIII site downstream of the of the Gy1promoter in pKS349 (SEQ ID NO:48).

Generation of Transgenic Somatic Embryos:

For co-expression of YL DGAT1 and YL DGAT2 gene in soybean somaticembryos soybean tissue was co-bombarded as described below with amixture of KS349 and KS362. Briefly, DNA of KS349 was digested withrestriction enzymes PstI, XhoI to inactivate the selectable marker genecassette (CaMV35S PRO/HPT/CaMV NOS TER). This DNA was mixed in a 10:1ratio with Sail-linearized plasmid DNA of KS362 and used for soybeantransformation as outlined below. Alternatively, soybean somatic embryossoybean tissue was bombarded as described below with intact plasmid DNAof KS364 which contains functional expression cassettes for both, YLDGAT1 and YL DGAT2. For expression of YL DGAT1 alone, uncut plasmid DNAof KS349 was used for particle bombardment of embryo tissue. Similarly,for expression of YL DGAT2 alone, uncut plasmid DNA of KS362 was usedfor particle bombardment of embryo tissue. Moreover, DNA that containeda selectable marker only (KS352) was used for soybean tissuetransformation in an identical fashion.

Culture Conditions:

Soybean embryogenic suspension cultures (cv. Jack) were maintained in 35mL liquid medium SB196 (infra) on a rotary shaker, 150 rpm, 26° C. withcool white fluorescent lights on 16:8 h day/night photoperiod at lightintensity of 60-85 μE/m2/s. Cultures were subcultured every 7 days totwo weeks by inoculating approximately 35 mg of tissue into 35 mL offresh liquid SB 196 (the preferred subculture interval is every 7 days).

Soybean embryogenic suspension cultures were transformed with thesoybean expression plasmids by the method of particle gun bombardment(Klein et al., Nature 327:70 (1987)) using a DuPont Biolistic PDS1000/HEinstrument (helium retrofit) for all transformations.

Soybean Embryogenic Suspension Culture Initiation:

Soybean cultures were initiated twice each month with 5-7 days betweeneach initiation. Pods with immature seeds from available soybean plants45-55 days after planting were picked, removed from their shells andplaced into a sterilized magenta box. The soybean seeds were sterilizedby shaking them for 15 min in a 5% Clorox solution with 1 drop of ivorysoap (i.e., 95 mL of autoclaved distilled water plus 5 mL Clorox and 1drop of soap, mixed well). Seeds were rinsed using 2 1-liter bottles ofsterile distilled water and those less than 4 mm were placed onindividual microscope slides. The small end of the seed was cut and thecotyledons pressed out of the seed coat. Cotyledons were transferred toplates containing SB199 medium (25-30 cotyledons per plate) for 2 weeks,then transferred to SB1 for 2-4 weeks. Plates were wrapped with fibertape. After this time, secondary embryos were cut and placed into SB196liquid media for 7 days.

Preparation of DNA for Bombardment:

Either an intact plasmid or a DNA plasmid fragment containing the genesof interest and the selectable marker gene were used for bombardment.

A 50 μL aliquot of sterile distilled water containing 1 mg of goldparticles was added to 5 μL of a 1 μg/μL DNA solution (either intactplasmid or DNA fragment prepared as described above), 50 μL 2.5M CaCl₂and 20 μL of 0.1 M spermidine. The mixture was pulsed 5 times on level 4of a vortex shaker and spun for 5 sec in a bench microfuge. After a washwith 150 μL of 100% ethanol, the pellet was suspended by sonication in85 μL of 100% ethanol. Five μL of DNA suspension was dispensed to eachflying disk of the Biolistic PDS1000/HE instrument disk. Each 5 μLaliquot contained approximately 0.058 mg gold particles per bombardment(i.e., per disk).

Tissue Preparation and Bombardment with DNA:

Approximately 100-150 mg of 7 day old embryonic suspension cultures wereplaced in an empty, sterile 60×15 mm petri dish and the dish was placedinside of an empty 150×25 mm Petri dish. Tissue was bombarded 1 shot perplate with membrane rupture pressure set at 650 PSI and the chamber wasevacuated to a vacuum of 27-28 inches of mercury. Tissue was placedapproximately 2.5 inches from the retaining/stopping screen.

Selection of Transformed Embryos:

Transformed embryos were selected using hygromycin as the selectablemarker. Specifically, following bombardment, the tissue was placed intofresh SB196 media and cultured as described above. Six to eight dayspost-bombardment, the SB196 is exchanged with fresh SB196 containing 30mg/L hygromycin. The selection media was refreshed weekly. Four to sixweeks post-selection, green, transformed tissue was observed growingfrom untransformed, necrotic embryogenic clusters. Isolated, greentissue was removed and inoculated into multi-well plates to generatenew, clonally propagated, transformed embryogenic suspension cultures.

Embryo Maturation:

Transformed embryogenic clusters were cultured for one-three weeks at26° C. in SB196 under cool white fluorescent (Phillips cool whiteEconowatt F40/CW/RS/EW) and Agro (Phillips F40 Agro) bulbs (40 watt) ona 16:8 hrphotoperiod with light intensity of 90-120 μE/m²s. After thistime embryo clusters were removed to a solid agar media, SB166, forlweek. Then subcultured to medium SB103 for 3 weeks. Alternatively,embryo clusters were removed to SB228 (SHaM) liquid media, 35 mL in 250mL Erlenmeyer flask, for 2-3 weeks. Tissue cultured in SB228 wasmaintained on a rotary shaker, 130 rpm, 26° C. with cool whitefluorescent lights on 16:8 h day/night photoperiod at light intensity of60-85 μE/m2/s. During this period, individual embryos were removed fromthe clusters and screened for alterations in their fatty acidcompositions as described supra.

Media Recipes:

SB 196 - FN Lite Liquid Proliferation Medium (per liter) MS FeEDTA -100x Stock 1 10 mL MS Sulfate - 100x Stock 2 10 mL FN Lite Halides -100x Stock 3 10 mL FN Lite P, B, Mo - 100x Stock 4 10 mL B5 vitamins (1mL/L) 1.0 mL 2,4-D (10 mg/L final concentration) 1.0 mL KNO₃ 2.83 gm(NH₄)₂SO₄ 0.463 gm Asparagine 1.0 gm Sucrose (1%) 10 gm pH 5.8 FN LiteStock Solutions Stock Number 1000 mL 500 mL 1 MS Fe EDTA 100x Stock Na₂EDTA* 3.724 g 1.862 g FeSO₄—7H₂O 2.784 g 1.392 g *Add first, dissolve indark bottle while stirring 2 MS Sulfate 100x stock MgSO₄—7H₂O 37.0 g18.5 g MnSO₄—H₂O 1.69 g 0.845 g ZnSO₄—7H₂O 0.86 g 0.43 g CuSO₄—5H₂O0.0025 g 0.00125 g 3 FN Lite Halides 100x Stock CaCl₂—2H₂O 30.0 g 15.0 gKI 0.083 g 0.0715 g CoCl₂—6H₂O 0.0025 g 0.00125 g 4 FN Lite P, B, Mo100x Stock KH₂PO₄ 18.5 g 9.25 g H₃BO₃ 0.62 g 0.31 g Na₂MoO₄—2H₂O 0.025 g0.0125 g SB1 Solid Medium (per liter) 1 package MS salts (Gibco/BRL -Cat. No. 11117-066) 1 mL B5 vitamins 1000X stock 31.5 g Glucose 2 mL2,4-D (20 mg/L final concentration) pH 5.7 8 g TC agar SB199 SolidMedium (per liter) 1 package MS salts (Gibco/BRL - Cat. No. 11117-066) 1mL B5 vitamins 1000X stock 30 g Sucrose 4 ml 2,4-D (40 mg/L finalconcentration) pH 7.0 2 gm Gelrite SB 166 Solid Medium (per liter) 1package MS salts (Gibco/BRL - Cat. No. 11117-066) 1 mL B5 vitamins 1000Xstock 60 g maltose 750 mg MgCl₂ hexahydrate 5 g Activated charcoal pH5.7 2 g Gelrite SB 103 Solid Medium (per liter) 1 package MS salts(Gibco/BRL - Cat. No. 11117-066) 1 mL B5 vitamins 1000X stock 60 gmaltose 750 mg MgCl2 hexahydrate pH 5.7 2 g Gelrite SB 71-4 Solid Medium(per liter) 1 bottle Gamborg's B5 salts w/ sucrose (Gibco/BRL - Cat. No.21153-036) pH 5.7 5 g TC agar 2,4-D Stock Obtain premade from PhytotechCat. No. D 295 - concentration 1 mg/mL B5 Vitamins Stock (per 100 mL)Store aliquots at −20° C. 10 g Myo-inositol 100 mg Nicotinic acid 100 mgPyridoxine HCl 1 g Thiamine

If the solution does not dissolve quickly enough, apply a low level ofheat via the hot stir plate.

SB 228 - Soybean Histodifferentiation & Maturation (SHaM) (per liter)DDI H2O 600 ml FN-Lite Macro Salts for SHaM 10X 100 ml MS Micro Salts1000x 1 ml MS FeEDTA 100x 10 ml CaCl 100x 6.82 ml B5 Vitamins 1000x 1 mlL-Methionine 0.149 g Sucrose 30 g Sorbitol 30 g Adjust volume to 900 mLpH 5.8 Autoclave Add to cooled media (≦30 C): *Glutamine (Final conc. 30mM) 4% 110 mL *Note: Final volume will be 1010 mL after glutamineaddition.Because glutamine degrades relatively rapidly, it may be preferable toadd immediately prior to using media. Expiration 2 weeks after glutamineis added; base media can be kept longer w/o glutamine.□

FN-lite Macro for SHAM 10X- Stock #1 (per liter) (NH₄)2SO₄ (AmmoniumSulfate) 4.63 g KNO₃ (Potassium Nitrate) 28.3 g MgSO₄*7H₂0 (MagnesiumSulfate Heptahydrate) 3.7 g KH₂PO₄ (Potassium Phosphate, Monobasic) 1.85g Bring to volume Autoclave MS Micro 1000X- Stock #2 (per 1 liter) H₃BO₃(Boric Acid) 6.2 g MnSO₄*H₂O (Manganese Sulfate Monohydrate) 16.9 gZnSO4*7H20 (Zinc Sulfate Heptahydrate) 8.6 g Na₂MoO₄*2H20 (SodiumMolybdate Dihydrate) 0.25 g CuSO₄*5H₂0 (Copper Sulfate Pentahydrate)0.025 g CoCl₂*6H₂0 (Cobalt Chloride Hexahydrate) 0.025 g KI (PotassiumIodide) 0.8300 g Bring to volume Autoclave FeEDTA 100X- Stock #3 (perliter) Na₂EDTA* (Sodium EDTA) 3.73 g FeSO₄*7H₂0 (Iron SulfateHeptahydrate) 2.78 g *EDTA must be completely dissolved before addingiron. Bring to Volume Solution is photosensitive. Bottle(s) should bewrapped in foil to omit light. Autoclave Ca 100X- Stock #4 (per liter)CaCl₂*2H₂0 (Calcium Chloride Dihydrate) 44 g Bring to Volume AutoclaveB5 Vitamin 1000X- Stock #5 (per liter) Thiamine*HCl 10 g Nicotinic Acid1 g Pyridoxine*HCl 1 g Myo-Inositol 100 g Bring to Volume Store frozen4% Glutamine- Stock #6 (per liter) DDI water heated to 30° C. 900 mlL-Glutamine 40 g Gradually add while stirring and applying low heat. Donot exceed 35° C. Bring to Volume Filter Sterilize Store frozen* *Note:Warm thawed stock in 31° C. bath to fully dissolve crystals.

Oil Analysis:

Somatic embryos were harvested after two weeks of culture in the liquidmaturation medium SB228 (SHaM) liquid media. Approximately 30 eventswere created in transformations with KS352, KS349/KS362, and KS362 andKS364. All embryos generated for a given event were harvested in bulkand processed as follows. Embryos were frozen on dry ice or byincubation in a −80° C. freezer for two h followed by lyophilization for48 h.

Dried embryos were ground to a fine powder using a genogrinder vial(½″×2″ polycarbonate) and a steel ball (SPEX Centriprep (Metuchen, N.J.,U.S.A.). Grinding time was 30 sec at 1450 oscillations per min. Forevery event, triplicates of approximately 10 mg of tissue were weighedinto Eppendorf tubes. The tissue was extracted using 200 μL heptane atroom temperature under continuous shaking for 2 h. Heptane extracts werecleared by centrifugation and 25 μL of extract was derivatized to fattyacid methyl esters as follows. One mL of a 25% sodium methoxide stocksolution was added to 24 mL of HPLC grade methanol. Sodium methoxide wasstored under an inert gas.

Five μL of a 17:0 TAG (Nu-Chek Prep, Elysian, Minn., USA) stock solution(10 mg/mL) was combined with 25 μL of heptane tissue extract in a glassculture tube 500 μL of 1% sodium methoxide was added. Sample werederivatized in a water bath at 50° C. for 15 min. Samples were allowedto cool to RT and 1 mL of 1M NaCl was added follwed by brief mixing.FAMEs were extracted into 1 mL of hepatene and 4 μL sample werequantitated by GC analysis.

Data analysis was performed by plotting the oleic content (% of totalFAME) against the total FAME content (% DW). TABLE 8 shows that somaticembryos generated with a vector control (KS352) show little fluctuationin oleic acid content and some fluctuation in oil content that can verylikely be attributed to biological variation that in introduced in theregeneration process. For example, embryos very likely show variation intheir developmental stage at the time of harvesting. In embryosgenerated with the control construct no correlation (R2=0.1142) wasobserved between the oleic acid content and the oil content (TABLE 8).In embryos generated with plasmid constructs expressing YL DGAT1 and YLDGAT2 s gene alone, KS349 and KS362, respectively or both YL DGAT1 andDGAT2 genes (KS349/KS362, KS364) under control of strong seed specificpromoters both oleic acid content and total esterified fatty acidcontent showed a wide range of fluctuation. Moreover, as shown in FIGS.4 and 5, a strong correlation (R²≧0.59) was observed between the oleicacid content and the total esterified fatty acid content for somaticembryos generated with KS349 and KS362 either alone or in combinationsas well as with KS364, a transformation plasmid that contains expressioncassettes for YL DGAT1 and YL DAT2 genes.

TABLE 8 Esterified Fatty Acid and Oleic Acid Content of Soybean SomaticEmbryos KS352 KS352 oleic acid oleic acid FAME (% total FAME (% totalEvent # (% DCW) stdv FAME) Event # (% DCW) stdv FAME) 22 6.2 nd 18.9 444.6 nd 17.1 16 5.7 nd 15.8 65 4.6 nd 19.4 35 5.6 nd 19.5 6 4.5 nd 13.948 5.6 nd 18.5 24 4.5 nd 16.1 14 5.5 nd 17.3 31 4.5 nd 15.9 43 5.5 nd18.7 20 4.4 nd 17.1 42 5.4 nd 19.3 37 4.4 nd 17.3 33 5.3 nd 17.2 69 4.4nd 19.2 68 5.3 nd 18.6 50 4.3 nd 17.2 3 5.2 nd 18.5 54 4.3 nd 19.5 4 5.2nd 18.9 55 4.3 nd 16.1 11 5.2 nd 19.1 64 4.3 nd 18.7 41 5.2 nd 16.9 324.1 nd 14.4 51 5.2 nd 18.2 61 4.1 nd 16.8 7 5.1 nd 17.2 23 4.0 nd 16.110 5.1 nd 19.9 26 4.0 nd 13.6 21 5.1 nd 18.2 49 4.0 nd 16.5 27 5.1 nd18.3 18 3.9 nd 16.4 1 5.0 nd 17.6 8 3.8 nd 15.5 46 5.0 nd 18.4 53 3.8 nd20.2 59 5.0 nd 17.5 63 3.8 nd 17.2 66 5.0 nd 19.3 52 3.7 nd 17.3 5 4.9nd 15.1 17 3.6 nd 14.3 15 4.9 nd 15.7 36 3.6 nd 15.7 29 4.9 nd 16.5 603.4 nd 16.6 2 4.8 nd 17.6 12 3.3 nd 15.4 9 4.8 nd 17.4 45 3.3 nd 15.2 304.8 nd 17.0 62 3.3 nd 18.8 34 4.8 nd 16.9 40 3.2 nd 13.3 19 4.7 nd 14.825 3.0 nd 12.3 47 4.7 nd 17.4 38 3.0 nd 16.2 67 4.7 nd 22.9 57 2.5 nd18.0 13 4.6 nd 17.2 56 2.3 nd 18.2 28 4.6 nd 15.9 58 2.2 nd 16.5 39 4.6nd 18.6 KS349 KS362 oleic acid oleic acid FAME (% total FAME (% totalEvent # (% DCW) stdv FAME) Event # (% DCW) stdv FAME) 28 10.8 1.0 33.228 14.7 0.3 28.9 16 10.6 3.1 32.7 16 14.5 0.1 33.1 25 10.3 0.9 34.1 1914.1 0.6 32.6 19 9.7 0.2 31.5 24 13.6 0.0 29.9 8 9.5 0.9 34.7 17 13.30.4 31.3 7 9.4 0.3 34.3 21 12.7 0.2 28.0 18 9.3 0.1 34.3 12 12.6 0.130.6 30 9.2 0.0 32.3 6 11.6 1.3 32.0 3 8.6 0.6 32.9 18 11.5 0.6 26.5 208.5 0.1 32.9 22 10.9 0.7 23.8 24 8.4 0.1 30.8 11 10.8 0.1 27.2 5 8.3 0.337.0 26 10.8 0.1 25.8 21 8.2 0.5 32.5 14 10.6 0.2 25.3 12 8.1 0.3 33.213 10.4 0.3 22.4 31 8.1 0.2 33.4 20 9.8 0.3 21.1 11 7.5 0.5 30.5 8 9.50.7 21.8 6 7.5 0.2 29.9 25 9.3 0.1 19.5 22 7.5 0.7 33.2 10 9.1 0.5 21.127 7.1 0.4 19.4 4 8.8 0.3 23.5 29 7.1 1.5 29.9 15 8.8 0.0 19.1 23 6.60.3 18.4 23 7.8 0.2 16.1 17 6.3 0.7 22.7 27 7.8 0.6 18.3 15 6.0 0.1 28.11 7.2 0.6 20.1 4 5.7 0.1 21.2 3 7.2 0.3 18.1 13 5.7 0.0 23.6 9 7.2 0.214.8 14 5.6 0.2 21.7 2 6.3 0.3 17.5 26 5.5 0.1 19.1 7 6.1 0.3 22.9 105.5 0.1 30.8 5 4.6 0.2 17.3 2 5.5 0.3 28.2 9 5.2 0.2 16.6 1 4.6 0.3 17.1KS349/KS362 KS364 oleic acid oleic acid FAME (% total FAME (% totalEvent # (% DCW) stdv FAME) Event # (% DCW) stdv FAME) 12 13.2 0.3 34.121 16.1 0.9 35.9 4 11.7 0.7 33.4 29 14.6 2.1 33.8 13 11.4 0.2 33.1 1814.2 2.3 33.2 18 11.1 0.2 33.5 27 13.4 0.8 31.7 24 11.1 0.1 33.3 20 12.20.8 35.6 3 10.9 0.0 34.1 28 11.8 0.7 34.6 10 10.8 0.2 31.8 26 11.5 1.630.8 9 10.7 0.2 33.5 3 11.3 1.0 36.2 23 10.6 0.4 32.7 22 10.9 1.4 34.017 10.2 0.8 31.5 1 10.7 0.6 30.8 29 10.0 0.5 26.6 24 10.7 0.6 31.2 119.9 0.3 31.3 25 10.6 0.2 31.2 16 9.4 0.1 31.7 6 10.2 0.4 32.6 19 9.3 0.328.1 2 10.1 0.3 31.0 1 8.9 0.5 27.4 5 9.9 0.5 33.3 25 8.5 0.5 31.8 119.8 0.4 37.8 7 8.4 0.0 17.6 12 9.8 0.6 37.0 15 8.4 0.1 29.8 8 9.4 0.131.5 26 8.3 0.2 18.6 10 9.3 0.1 31.2 6 8.1 0.2 24.5 15 9.2 0.4 33.7 57.4 0.1 19.3 16 9.1 0.2 33.9 21 7.4 0.8 18.0 19 8.7 0.1 27.3 27 6.9 0.825.7 7 8.4 0.6 26.7 30 6.9 0.7 24.7 4 7.6 0.1 28.1 2 6.0 0.3 18.2 23 5.70.2 21.1 14 5.6 0.4 23.0 9 4.2 0.3 15.1 22 5.6 0.2 17.6 14 4.2 0.2 15.18 4.6 0.1 17.4 17 3.6 0.4 21.9 28 4.2 0.2 19.7 13 3.5 0.1 15.0 20 2.80.1 11.9

In summary, the data shows that in soybean somatic embryos, similar toArabidopsis seed YL DGAT gene expression is associated with increasedincorporation of oleic acid into the total esterified fatty acidfraction. However, in contrast to Arabidopsis, in soybean somaticembryos increased oleic acid content is tightly correlated with totalaccumulation of esterified fatty acid. In other words, expression of YLDGAT2 alone and co-expression of YL DGAT1 and YL DAGT2 in soybeansomatic embryos leads to increased biosynthesis and incorporation offatty acids into the total esterified fatty acid fraction. Takentogether this finding strongly suggests that expression of YL DGAT genesprovides an efficient strategy to achieve an increase in the total ofoil content of soybean seed.

Example 6 Expression of Yarrowia lipolytica DGAT Genes in Soybean Seed

Construction of a control plasmid (KS332) (FIG. 2) containing only CaMV35S PRO/HPT/NOS TER and Kti PRO/DsRed/Kti TER expression cassettes isdescribed in Example 5. Its sequence is set forth as SEQ ID NO:51.

Transgenic soybean lines were generated by the method of particle gunbombardment (Klein et al., Nature (London) 327:70-73 (1987); U.S. Pat.No. 4,945,050) using a BIORAD Biolistic PDS1000/He instrument andplasmid DNA of KS332, KS362 and a 10:1 mixture of KS349 and KS362prepared as described in Example 3. The following stock solutions andmedia were used for transformation and regeneration of soybean plants:

Stock solutions:

Sulfate 100× Stock:

-   -   37.0 g MgSO₄.7H₂O, 1.69 g MnSO₄.H₂O, 0.86 g ZnSO₄.7H₂O, 0.0025 g        CUSO₄. 5H₂O

Halides 100× Stock:

30.0 g CaCl₂.2H₂O, 0.083 g KI, 0.0025 g CoCl₂.6H₂O

P, B, Mo 100× Stock:

18.5 g KH₂PO₄, 0.62 g H₃BO₃, 0.025 g Na₂MoO₄.2H₂O

Fe EDTA 100× Stock:

3.724 g Na₂EDTA, 2.784 g FeSO₄.7H₂O

2,4-D Stock:

-   -   10 mg/mL Vitamin B5 1000X Stock: 10.0 g myo-inositol, 0.10 g        nicotinic acid, 0.10 g pyridoxine HCl, 1 g thiamine.

Media (per Liter):

-   -   SB196: 10 mL of each of the above stock solutions, 1 mL B5        Vitamin stock, 0.463 g (NH₄)₂ SO₄, 2.83 g KNO₃, 1 mL 2,4-D        stock, 1 g asparagine, 10 g Sucrose, pH 5.7

SB103:

-   -   1 pk. Murashige & Skoog salts mixture, 1 mL B5 Vitamin stock,        750 mg MgCl₂ hexahydrate, 60 g maltose, 2 g gelrite, pH 5.7.

SB166:

SB103 supplemented with 5 g per liter activated charcoal.

SB71-4:

Gamborg's B5 salts, 1 mL B5 vitamin stock, 30 g sucrose, 5 g TC agar, pH5.7.

To prepare tissue for transformation, soybean embryogenic suspensioncultures were maintained in 35 mL liquid medium (SB196) on a rotaryshaker (150 rpm) at 28° C. with fluorescent lights providing a 16 hday/8 h night cycle. Cultures were subcultured every two weeks byinoculating approximately 35 mg of tissue into 35 mL of fresh liquidmedia.

In particle gun bombardment procedures it is possible to use purified 1)entire plasmid DNA; or 2) DNA fragments containing only the recombinantDNA expression cassette(s) of interest. For every seventeen bombardmenttransformations, 85 μL of suspension is prepared containing 1 to 90picograms (pg) of plasmid DNA per base pair of each DNA plasmid. Bothrecombinant DNA plasmids were co-precipitated onto gold particles asfollows. The DNAs in suspension were added to 50 μL of a 20-60 mg/mL 0.6μm gold particle suspension and then combined with 50 μL CaCl₂ (2.5 M)and 20 μL spermidine (0.1 M). The mixture was vortexed for 5 sec, spunin a microfuge for 5 sec, and the supernatant removed. The DNA-coatedparticles were then washed once with 150 μL of 100% ethanol, vortexedand spun in a microfuge again, then resuspended in 85 μL of anhydrousethanol. Five μL of the DNA-coated gold particles were then loaded oneach macrocarrier disk.

Approximately 150 to 250 mg of two-week-old suspension culture wasplaced in an empty 60 mm×15 mm petri plate and the residual liquidremoved from the tissue using a pipette. The tissue was placed about 3.5inches away from the retaining screen and each plate of tissue wasbombarded once. Membrane rupture pressure was set at 650 psi and thechamber was evacuated to −28 inches of Hg. Three plates were bombarded,and, following bombardment, the tissue from each plate was dividedbetween two flasks, placed back into liquid media, and cultured asdescribed above.

Seven days after bombardment, the liquid medium was exchanged with freshSB196 medium supplemented with 30-50 mg/L hygromycin. The selectivemedium was subsequently refreshed weekly or biweekly. Seven weekspost-bombardment, bright green, transformed tissue was observed growingfrom untransformed, chlorotic or necrotic embryogenic clusters. Isolatedgreen tissue was removed and inoculated into individual wells insix-well culture dishes to generate new, clonally-propagated,transformed embryogenic suspension cultures. Thus, each new line wastreated as independent transformation event in an individual well. Thesesuspensions can then be maintained as suspensions of embryos clusteredin an immature developmental stage through subculture or they can beregenerated into whole plants by maturation and germination ofindividual somatic embryos.

After two weeks in individual cell wells, transformed embryogenicclusters were removed from liquid culture and placed on solidifiedmedium (SB166) containing no hormones or antibiotics for one week.Embryos were cultured for at 26° C. with mixed fluorescent andincandescent lights on a 16 h day/8 h night schedule. After one week,the cultures were then transferred to SB103 medium and maintained in thesame growth conditions for 3 additional weeks.

Somatic embryos became suitable for germination after four weeks andwere then removed from the maturation medium and dried in empty petridishes for one to five days. The dried embryos were then planted inSB71-4 medium where they were allowed to germinate under the same lightand temperature conditions as described above. Germinated embryos weretransferred to sterile soil and grown to maturity for seed production.

A total of 29 transgenic lines with seed were generated with intactplasmid DNA of KS362 at concentration of 15 pg per by of plasmid DNA pergold particle preparation (see above). For every event 20 seed werescored for the presence of the DS marker gene. Briefly, seeds wereobserved under a stereo microscope (Leica MZ Fluo III) using a UV lightsource. A filter set customized for fluorescence associated with DsRedexpression with the follwing properties was used: Exitation λ=540-580nm/Emssion λ≧570 nm. In cases were less that 20 seed were available allseed were scored in this manner. Subsequently soybean seed oil contentwas measured by NMR as described previously 3. Nineteen events generatedwith KS362 contained seed that were positive for DsRed. Of these, 11events showed a detectable difference in oil content between DsRedpositive transgenic segregants and DsRed negative null segregants. Dataare summarized in TABLE 9.

TABLE 9 Oil Content of T1 Soybean Seed Generated with KS362 avg % avg %oil oil delta % n null n DsRed+ points delta % AFS4822.4.5.1 5 13.3 1520.4 7.2 54.1 AFS4822.3.2.1 10 14.2 9 18.9 4.7 32.7 AFS4822.3.3.1 5 12.48 16.1 3.7 29.5 AFS4822.4.2.1 4 16.2 6 20.9 4.7 29.3 AFS4822.4.1.1 915.1 8 19.4 4.3 28.9 AFS4822.1.13.1 5 15.3 15 19.1 3.7 24.4AFS4822.1.2.1 2 15.8 18 19.6 3.8 24.1 AFS4822.4.17.1 7 14.9 13 18.5 3.623.9 AFS4822.1.9.1 8 16.9 12 19.8 3.0 17.5 AFS4822.2.11.1 6 17.9 14 20.72.8 15.7 AFS4822.2.10.1 6 20.6 14 23.5 2.8 13.7

A total of 10 transgenic lines with seed were generated with DNA ofKS332. For every event 20 seed were scored for the presence of the DSmarker gene as described above. In cases were less that 20 seed wereavailable all seed were scored in this manner. Subsequently soybean seedoil content was measured by NMR as described in Example 4. Seven eventsgenerated with KS 332 contained seed that were positive for DsRed. Dataare summarized in TABLE 10.

TABLE 10 Oil Content of T1 Soybean Seed Generated with KS332 avg % avg %oil oil delta % n null n DsRed+ points delta % AFS4703.1.1.1 3 21.8 1720.5 −1.4 −6.2 AFS4703.1.2.1 4 20.8 16 18.5 −2.3 −11.2 AFS4703.1.6.1 520.4 15 21.4 1.0 5.0 AFS4703.2.3.1 8 22.1 12 21.1 −0.9 −4.1AFS4703.2.4.1 6 22.2 14 21.7 −0.4 −2.0 AFS4703.3.8.1 6 21.8 14 21.8 0.00.2 AFS4703.3.16.1 5 23.9 15 23.3 −0.6 −2.5

In contrast to seed generated with KS362, for transgenic seed generatedwith KS332 no consistent oil increase could be associated with thepresence of the DsRed marker in T1 segregants.

Four events generated with KS362 were subjected to analysis of DsRedstatus and oil NMR of all available T1 seed. Data are summarized inTABLE 11.

TABLE 11 Oil Content of T1 Soybean Seed Generated with KS362 avg % avg %oil oil delta % n null n DS red+ points delta % AFS4822.4.5.1 8 12.0 2418.3 6.3 52.9 AFS4822.1.13.1 17 15.0 42 18.7 3.7 24.6 AFS4822.1.2.1 415.7 40 19.3 3.5 22.3 AFS4822.2.10.1 8 19.5 25 23.0 3.5 17.8AFS4822.2.11.1 6 17.9 17 20.3 2.4 13.5

In summary, the data in the previous tables and FIG. 7 show that seedspecific expression of YL DAGT2 leads to increased oil biosynthesisduring soybean seed maturation and thus provides an efficient metabolicengineering tool to increase oil accumulation in soybeans.

A total of 16 transgenic lines with seed were generated byco-bombardment with DNA of KS349 and KS362 that had been mixed at a 10:1ratio (see Example 4). The DNA mixture was delivered in soybeantransformation at a final concentration of 15 pg per by of plasmid DNAper gold particle preparation. Briefly, prior to bombardment DNA KS349was digested with PstI, XhoI for inactivation of the selectable markergene on this plasmid. DNA of KS362 was linearized with SalI. It isreasonable to assume that because of the pre-treament of the DNA, theselectable marker gene in all transformations was delivered from theplasmid (KS362) that was bombarded at the lower DNA concentration.Initial inspection of these seed under the fluorescencestereo-microcsope revealed that very few events of this transformationcontained T1 seed that were positive for the DsRed marker gene. Thisresult may be due to the proximity of the DsRed expression cassette tothe end of the SalI restriction fragment of KS362 that was used forsoybean transformation. It may have resulted in the integration of KS362DNA fragments that did not contain a functional DsRed expressioncassette. For this reason T1 seed were screened for the absence ofpresence of the transgene-derived YL DGAT by assaying the seed fattyacid composition. For every event 20 seed were analyzed by GC. 50 seedof untransformed soybean seed were processed in the same manner. Soybeanseed chips were produced by cutting the seed with a razorblade avoidingthe embryonic axis. Seed chips of approximately 2 mg were placed in avial containing 50 μL trimethylsulfonium hydroxide and 0.5 mL hexane.The chips were incubated for 30 min at room temperature while shaking. 5μL of the hexane layer was injected into a Hewlett Packard 6890 GasChromatograph containing a Omegawax 320 fused silica capillary column(Supelco Cat. No. 24152). Oven conditions were as follows: initialtemperature of 220° C. for 2.7 minutes, ramped to 240° C. over 1 min andheld at 240° C. For a total run time of 6 min. Retention times werecompared to standards commercially available (Nu-Chek Prep, Inc. Cat.No. U-99-A). Fatty acids were determined by direct trans-esterificationof individual standards in 0.5 mL of methanolic H2504 (2.5%). Fatty acidmethyl esters were extracted from the methanolic solutions into hexaneafter the addition of an equal volume of water.

Ten events were identified that contained T1 seed with ≧25% oleic acidcontent. Since this oleic acid content was not observed in untransformedsoybean seed (see FIG. 6 and TABLE 12) and increased oleic acid contentwas previously associated with YL DGAT2 and YL DGAT1 and YL DGAT2co-expression both in arabidopsis seed and soybean somatic embryos, itis believed that the presence of oleic acid at levels of ≧25% providesefficient means to identify YL DGAT positive T1 seed. After GC analysisfor YL DGAT genotyping, seed were subjected to oil measurements by NMRas described previously (Example 3). When oleic acid content was plottedagainst total oil content seven of the 10 events with T1 seed of ≧25%showed a correlation of R²≧0.3 between oil and oleic acid content. Theproperties of these events are described in more detail in TABLE 12.

TABLE 12 Oil Content of T1 Soybean Seed Generated with KS349/KS362 avg %avg % oil <25% oil ≧25% delta % R² % n oleic n oleic points delta %oleic/% oil AFS4818.3.1.1 15 11.9 5 16.8 4.9 41.0 0.38 AFS4818.1.5.1 1015.5 10 20.0 4.5 28.7 0.69 AFS4818.2.10.1 10 13.2 10 16.5 3.3 25.2 0.39AFS4818.1.9.1 5 12.9 14 15.9 3.0 22.9 0.51 AFS4818.2.6.1 12 18.6 8 21.93.4 18.1 0.51 AFS4818.1.3.1 6 20.5 14 23.6 3.1 14.9 0.45 AFS4818.1.2.1 619.4 14 21.8 2.4 12.4 0.37 Jack (wt control) 56 21.7 0.003

Jack wild type seed were grown under similar condition to those used forT1 seed generation and analyzed by GC and NMR analysis. It was observedthat oleic acid and oil content fluctuated between 7.6-20.5% and18.6-25.2%, respectively. No correlation between oleic acid content andoil content could be observed in untransformed soybean seed.

Four events generated with KS349/KS362 were subjected to GC and NMRanalysis of all available T1 seed. Data are summarized in TABLE 13.

TABLE 13 Oil Content of T1 Soybean Seed Generated with KS349/KS362 avg %avg % oil <25% oil ≧25% delta % R² % n oleic n oleic points delta %oleic/% oil AFS4818.1.5.1 21 16.3 21 20.2 3.9 23.8 0.55 AFS4818.2.6.1 4918.1 23 21.9 3.7 20.7 0.50 AFS4818.1.3.1 33 20.2 67 23.5 3.3 16.2 0.43AFS4818.1.2.1 13 19.3 26 22.1 2.7 14.1 0.45

Taken together the data in the previous tables and FIGS. 6 and 7strongly support the conclusion that co-expression of YL DGAT1 and YLDGAT2 genes, like expression of the YL DGAT2 gene alone, provides anefficient strategy to achieve an increase in the total of oil content ofsoybean seed. Additionally, it should be noted that a high number ofevents could be identified with an oil difference of ≧2% points betweennull and transgenic segregants among a small set of transgenic eventsscreened.

Example 7 Expression of Yarrowia lipolytica DGAT Genes in Maize

Based on results disclosed in Examples 4, 5 and 6 of the instantapplication, the YL DGAT1 and YL DGAT2 genes can be expressed in theseed embryo of maize to increase the oil content of this tissue. Asdescribed below, this result can be achieved by transforming maize withexpression cassettes comprising open reading frames of DGAT1 and DGAT2from Yarrowia lipolytica operably linked on their 5′ ends to embryopreferred promoters, such as the promoter for the maize 16 kDa oleosingene (Lee, K. and Huang, A. H., Plant Mol. Biol. 26:1981-1987 (1984))and maize embryo abundant (EAP1) promoter and terminator (US2006272058A1).

An expression cassette comprising the promoter from the maize 16 kDaoleosin gene (OLE PRO), the coding sequence of the YL DGAT2 gene (SEQ IDNO:9) and the polyadenylation signal sequence/terminator from thenopaline synthase (NOS) gene of Agrobacterium tumefaciens is constructedusing methods and technologies known in the art. A second expressioncassette comprises the YL DGAT1 gene under the transcriptional controlof the maize embryo abundant protein (EAP1) promoter and terminator,with the maize ADH1 INTRON1 inserted between the promoter and codingsequence for enhanced expression. The two expression cassettes arelinked, together with a gene encoding a selectable marker, in a binaryvector suitable for Agrobacterium-mediated transformation of maize.

An Agrobacterium-based protocol can be used for the transformation ofmaize (see below). The resulting binary vector is introduced intoAgrobacterium LBA4404 (PHP 10523) cells, preferably by electroporation.An in vivo recombination generates a cointegrate plasmid between theintroduced binary vector and the vir plasmid (PHP10523) resident in theAgrobacterium cells. The resulting Agrobacterium cells are used totransform maize.

Transformation of Maize Mediated by Agrobacterium:

Freshly isolated immature embryos of maize, about ten days afterpollination (DAP), can be incubated with the Agrobacterium. Thepreferred genotype for transformation is the highly transformablegenotype Hi-II (Armstrong, Maize Gen. Coop. Newsletter 65:92-93 (1991)).An F1 hybrid created by crossing a Hi-II with an elite inbred may alsobe used. After Agrobacterium treatment of immature embryos, the embryoscan be cultured on medium containing toxic levels of herbicide. Onlythose cells that receive the herbicide resistance gene, and the linkedgene(s), grow on selective medium. Transgenic events so selected can bepropagated and regenerated to whole plants, produce seed, and transmittransgenes to progeny.

Preparation of Agrobacterium:

The engineered Agrobacterium tumefaciens LBA4404 can be constructed tocontain plasmids for seed-preferred expression of YL DGAT1 and YL DGAT2genes, as disclosed in U.S. Pat. No. 5,591,616 (the contents of whichare hereby incorporated by reference). To use the engineered constructin plant transformation, a master plate of a single bacterial colonytransformed with plasmids for seed-preferred expression of YL DGAT1 andYL DGAT2 genes can be prepared by inoculating the bacteria on minimal ABmedium and allowing incubation at 28° C. for approximately three days.(The composition and preparation of minimal AB medium has beenpreviously described in PCT Publication No. WO 02/009040 (the contentsof which are hereby incorporated by reference). A working plate can thenbe prepared by streaking the transformed Agrobacterium on YP medium(0.5% (w/v) yeast extract, 1% (w/v) peptone, 0.5% (w/v) sodium chloride,1.5% (w/v) agar) that contains 50 μg/mL of spectinomycin.

The transformed Agrobacterium for plant transfection and co-cultivationcan then be prepared one day prior to maize transformation. Into 30 mLof minimal A medium (prepared as described in PCT Publication No. WO02/009040) in a flask was placed 50 μg/mL spectinomycin, 100 μMacetosyringone, and about a ⅛ loopful of Agrobacterium from a one totwo-day-old working plate. The Agrobacterium can then be grown at 28° C.with shaking at 200 rpm for approximately fourteen h. At mid-log phase,the Agrobacterium can be harvested and resuspended at a density of 3 to5×108 CFU/mL in 561Q medium that contains100 μM acetosyringone usingstandard microbial techniques. The composition and preparation of 561Qmedium was described in PCT Publication No. WO 02/009040.

Immature Embryo Preparation:

Nine to ten days after controlled pollination of a maize plant,developing immature embryos are opaque and 1-1.5 mm long. This length isthe optimal size for infection with the PHP18749-transformedAgrobacterium. The husked ears can be sterilized in 50% commercialbleach and one drop Tween-20 for thirty minutes, and then rinsed twicewith sterile water. The immature embryos can then be aseptically removedfrom the caryopsis and placed into 2 mL of sterile holding solutionconsisting of medium 561Q that contains 100 μM of acetosyringone.

Agrobacterium Infection and Co-cultivation of Embryos:

The holding solution can be decanted from the excised immature embryosand replaced with transformed Agrobacterium. Following gentle mixing andincubation for about five minutes, the Agrobacterium can be decantedfrom the immature embryos. Immature embryos were then moved to a plateof 562P medium, the composition of which has been previously describedin PCT Publication No. WO 02/009040. The immature embryos can be placedon this media scutellum surface pointed upwards and then incubated at20° C. for three days in darkness. This step can be followed byincubation at 28° C. for three days in darkness on medium 562P thatcontains 100 μg/mL carbenecillin as described in U.S. Pat. No.5,981,840.

Selection of Transgenic Events:

Following incubation, the immature embryos can be transferred to 5630medium, which can be prepared as described in PCT Publication No. WO02/009040. This medium contains Bialaphos for selection of transgenicplant cells as conferred by the BAR gene that is linked to barley HGGTexpression cassette. At ten to fourteen-day intervals, embryos weretransferred to 5630 medium. Actively growing putative transgenicembryogenic tissue can be after six to eight weeks of incubation on the5630 medium.

Regeneration of T₀ Plants:

Transgenic embryogenic tissue is transferred to 288W medium andincubated at 28° C. in darkness until somatic embryos matured, or aboutten to eighteen days. Individual matured somatic embryos withwell-defined scutellum and coleoptile are transferred to 272 embryogermination medium and incubated at 28° C. in the light. After shootsand roots emerge, individual plants are potted in soil and hardened-offusing typical horticultural methods.

288W medium contains the following ingredients: 950 mL of deionizedwater; 4.3 g of MS Salts (Gibco); 0.1 g of myo-inositol; 5 mL of MSVitamins Stock Solution (Gibco); 1 mL of zeatin (5 mg/mL solution); 60 gsucrose; 8 g of agar (Sigma A-7049, Purified), 2 mL of indole aceticacid (0.5 mg/mL solution*); 1 mL of 0.1 mM ABA*; 3 mL of Bialaphos (1mg/mL solution*); and 2 mL of carbenicillin (50 mg/mL solution). The pHof this solution is adjusted to pH 5.6. The solution is autoclaved andingredients marked with an asterisk (*) are added after the media hascooled to 60° C.

Medium 272 contains the following ingredients: 950 mL of deionizedwater; 4.3 g of MS salts (Gibco); 0.1 g of myo-inositol; 5 mL of MSvitamins stock solution (Gibco); 40 g of Sucrose; and 1.5 g of Gelrite.This solution is adjusted to pH 5.6 and then autoclaved.

Example 8 Analysis of Kernel Oil Content Nuclear Magnetic Resonance(NMR) Analysis:

Seed are imbibed in distilled water for 12-24 hours at 4° C. The embryois dissected away and stored in a 48 well plate. The samples arelyophilized over-night in a Virtis 24×48 lyophilizer. The NMR (ProcessControl Technologies—PCT (Ft. Collins, Colo.) is set up as per themanufacturer's instructions. The NMR is calibrated using a series of 5mm NMR tubes containing precisely measured amounts of corn oil (Mazola).The calibration standards are 3, 6, 9, 12, 15, 18, 21, 27, 33, and 40 mgof oil.

Example 9 Synthesis of YL DGAT1 and YL DGAT2 genes

Nucleotide sequences encoding YL DGAT1 and YL DGAT2 were designed foroptimized expression in soybean seed using methods similar to thosedescribed in Wu, G et al. Nucleic Acids Research (2007), 35: D76-D79;Villalobos, A. et al. BMC Bioinformatics (2006), 7 No pp. given; Wu, G.et al. Protein Expression and Purification (2006), 47: 441-445;Richardson, S. M. et al. Genome Research (2006), 16: 550-556; Jayaraj,S. et al. Nucleic Acids Research (2005) 33: 3011-3016. DNA moleculeswere synthezised by DNA 2.0 (Menlo Park, Calif., USA).Expression-optimized DNA sequences of YL DGAT 1 and YL DGAT2 are setforth in SEQ ID NO:64 and SEQ ID NO:66, respectively. The amino acidsequences for soy optimized enzymes are set forth in SEQ ID NO:65 (YLDGAT1) and SEQ ID NO:67 (YL DGAT2) and are identical to the translationproducts of SEQ ID NO:1 and SEQ ID NO:9, respectively.

Example 10 Fatty Acid Composition of Soybean Somatic Embryos ExpressingYL DGAT Genes

Transgenic somatic embryos were generated using the plasmid constructsKS352, KS349, KS362 and KS364. Generation of the DNA constructs and thetransformation process is described in detail in EXAMPLE 5. Fatty acidcomposition was determined by GC analysis of fatty acid methyl estersgenerated by sodium methoxide derivatization of heptane extracts. Thefindings are summarized in TABLE 14. The table compares the fatty acidcomposition of 100 events generated with a control plasmid lacking YLDGAT genes with that of events created with plasmids containing YL DGAT1(KS349), YL DGAT2 (KS362) or both genes (KS364). For events generatedwith YL DGAT containing DNA constructs the average fatty acidcomposition of all events with greater than 30% oleic is shown.

TABLE 14 Fatty Acid Composition of Soybean Somatic Embryos generatedwith KS 352, 349, 362, 349&362 and 364 palmitic stearic oleic linoleiclinolenic Plasmid n acid acid acid acid acid KS352 average 100 15.9 5.217.9 44.1 16.9  KS352 range 12.6-20.8 4.2-6.6 12.3-22.9 39.3-46.912.4-23.5 KS349 average 18 11.6 5.4 33.0 41.3 8.6 (>30% oleic) KS349range 10.7-12.8 4.2-6.5 30.5-37.0 38.7-44.6  7.8-10.7 (>30% oleic) KS362average 5 11.5 6.3 31.9 43.2 6.9 (>30% oleic) KS362 range 10.9-12.75.7-7.0 30.6-33.1 41.9-44.8 6.2-7.7 (>30% oleic) KS349&362 14 12.8 5.433.8 39.7 8.4 average (>30% oleic) KS349&362 11.5-14.4 3.8-7.0 30.8-35.5  38-42.7  6.3-10.3 range (>30% oleic) KS364 average 14 10.9 6.4 39.038   5.7 (>30% oleic) KS364 range  9.2-12.6 5.9-7.7 32.8-48.6 31.4-42.73.2-6.5 (>30% oleic)

The table shows that expression of YL DGAT1 or YL DGAT2 as well asco-expression of said genes alters the FA profile of soybean somaticembryos. The most pronounced alteration is an increase in oleic acid anda decrease in linolenic acid that is consistently observed with all DNAconstructs tested. Expression of YL DGAT genes also leads to a decreasein palmitic and linoleic acid and an increase in stearic acid.

Example 11 Fatty Acid Composition of Soybean Seed Expressing YL DGATGenes

Event AFS4822.1.13.1 was generated using plasmid DNA of KS362 asdescribed in Example 6. Transgenic T1 seed show an increase in oilcontent of 24.6% when compared to null segregant seed from the same T1plant. This observation strongly supports the conclusion that thisevents expresses YL DGAT2. T1 seed of AFS4822.1.13.1 with or without theYL DGAT2 transgene were germinated and grown in the growth chamber forthree month. DS-red positive T1 seed of event AFS4703.1.6 weregerminated and grown alongside the YL DGAT2 event. AFS4703.1.6 wasgenerated with KS332, a plasmid vector hat contains the DS-red markergene but does not contain YL DGAT2 (see EXAMPLE 5). Several T1segregants could be identified that only produced DS-red positive T2seed indicating that these lines were homozygous for the respectivetransgene. T2 seed harvested from null-segregant progeny were DS-rednegative confirming that these lines likely did not contain a functionaltransgene.

For each selection six seed were chipped and the fatty acid compositionof the seed chips was analyzed by TMSH-derivatization followed by gaschromatography as described in EXAMPLE 6. TABLE15A compares the averagefatty acid composition of six seed chips of DS-red positive segregantsof AFS4822.1.13.1 with that of seed chips derived form a null-segregantplant and that of seed chips of event AFS4703.1.6 containing only theDS-red marker gene. It demonstrates that expression of YL DGAT2 altersthe FA profile of soybean seed. The most pronounced alteration is anincrease in oleic acid and a decrease in linolenic acid. Expression ofYL DGAT genes also leads to a decrease in palmitic and linoleic acid andan increase in stearic acid.

TABLE 15A Fatty Acid Composition of T2 Soybean Seed Generated with KS362palmitic stearic oleic linoleic linolenic Event n acid acid acid acidacid AFS4822.1.13.1 6 10.8 4.6 29.2 49.5 6.0 average (DS red positive)AFS4822.1.13.1 6 12.0 3.4 16.3 59.0 9.4 average (Null segregant)AFS4703.1.6 6 11.5 3.3 15.8 59.8 9.6 average (DS red positive)

Events AFS4818.1.9 and AFS4818.1.3 were generated using a mixture of DNAfragments derived from plasmids KS349 (YL DGAT1) and KS362 (YL DGAT2) asdescribed in EXAMPLE 5. Transgenic T1 seed of these two events show anincrease in oil content of 22.9 and 16.2%, respectively when compared tonull segregant seed from the same T1 plant. Although this observationstrongly supports the conclusion that both events express transgenederived YL DGAT it is not clear if both events contain intact copies ofboth or just one DGAT gene present in the DNA mixture used fortransformation. T1 seed with increased oil and oleic acid content (seeExample 6) were planted for events AFS4818.1.2, AFS4818.1.3,AFS4818.1.5, AFS48182.6, AFS4818.1.9. DNA was isolated, digested withthe two restriction enzymes EcoRI and HindIII and transferred to nylonmembranes using standard protocols. Duplicate blots were produced andhybridized independently with probes corresponding to a 1.21 kbrestriction fragment of the YL DGAT1 gene (generated by digestion ofKS349 with NcoI/EcoRI) and the intact YL DGAT2 genes (generated by NotIdigestion of KS 362). Based on the sequence of KS 349 (SEQ ID NO:48) andKS 362 (SEQ ID NO: 52) insertion of an intact copy of YL DGAT1 and YLDGAT2 gene in the soybean genome would be indicated by a stronghybridization signal of restriction fragments with a size of ≧1.908 and≧3.335 kb, respectively. In keeping with this, all events showedstrongly hybridizing bands of ≧1.908 kb when a YL DGAT 1 probe was used(FIG. 8 A). No hybridization signal was observed when DNA fromunmodified soybeans was used (lanes 11 and 12, FIGS. 8A and B). Thisdemonstrates that all events tested have insertions of at least one copyof the intact YL DGAT1 expression cassette present on KS 349. Howeverwhen DNA of YL DGAT2 was used in hybridization experiments, event4818.1.9 did only show a very weakly hybridizing band of high MW whereasall other events tested showed strongly hybridizing bands of ≧3.335 kb(lane 9, FIG. 8 B). Next genomic DNA of all five events was digestedwith BstXI, transferred to nylon membranes and probed with intact YLDGAT2 DNA generated as described above (FIG. 9). Insertion of an intactcopy of the YL DGAT2 expression cassette would be indicated by stronglyhybridizing bands of 0.584 kb (internal fragment) and additionalfragments of ≧0.2 and ≧0.77 kb. All events except 4818.1.9 show thehybridization pattern indicative of complete insertion of YL DGAT2. Itwas concluded that 4818.1.9 only contains a functional expression unitfor YL DGAT1.

T1 plants of events 4818.1.9 and 4818.1.3 that were derived from seedwith increased oil and oleic content of event 4818.1.9 and 4818.1.3 weregrown to maturity and seed were harvested. Fatty acid composition of T2seed was determined by TMSH-derivatization and GC analysis of seed chipsderived from T2 seed. Table compares fatty acid composition oftransgenic and null segregant seed from a T2 plant of 4818.1.9 and4818.1.3 (Table 15B). It demonstrates that expression of YL DGAT1 aswell as co-expression of YL DGAT1 and YL DGAT2 alters the FA profile ofsoybean seed. The most pronounced alteration is an increase in oleicacid and a decrease in linolenic acid. Expression of YL DGAT genes alsoleads to a decrease in palmitic and linoleic acid and an increase instearic acid.

TABLE 15B Fatty Acid Composition of T2 Soybean Seed Generated byco-transformation with KS349 and KS362 palmitic stearic oleic linoleiclinolenic Event n acid acid acid acid acid AFS4818.1.9 58 10.8 4.5 27.949.6 7.2 average AFS4818.1.9 42 12.2 3.4 14.3 57.0 13.0 average (Nullsegregant) AFS4818.1.3 34 10 4.2 31.6 48.7 5.5 average AFS4818.1.3 1411.4 3.0 16.4 58.7 10.5 average (Null segregant)

Example 12 Analysis of transgenic events: Growth Chamber

The present example describes measurements of oil content of soybeanderived form T2 plants that were homozygous or heterozygous fortransgenes comprising YL DGAT1 or YL DGAT2 or both YL DGAT genes. T2plants were grown in a controlled environment (growth chamber).

Oil analysis of T2 soybean seed derived from plants grown in a plantgrowth chamber was performed by NMR. Seed were harvested form individualplants. Seed selections from heterozygous plants derived formtransformations with the DGAT2 gene from Yarrowia showed segregation ofthe DS red marker. Oil content of DS red positive seed (with DGAT2transgene) and null segregant seed from the same plant is shown in Table16. In said table oil content of seed containing the DGAT2 transgene iscompared to that of non-transgenic null segregant seed from the sameplant.

TABLE 16 Oil content of transgenic seed and null segregant seed derivedfrom transgenic soybean T2 plants that segregate for transgene with theyarrowia DGAT2 gene average average oil (%) oil (%) Δ % Event PLANT wtransg. n null n points Δ % 4822.1.13 A 22.2 37 19.6 11 2.3 11.5 B 21.427 19.5 13 average 21.8 19.6 4822.4.5 A 22.3 35 18.4 13 3.6 19.6 B 22.538 18.5 10 C 21.7 31 17.7 17 D 22.7 31 18.2 17 E 21.7 23 18.9 17 average22.2 18.6 4822.1.9 A 22.9 33 20.4 7 2.5 12.3 4822.2.10 A 24.3 62 20.1 243.3 16.3 B 23 30 19.5 10 C 22.5 23 20.5 17 average 23.3 20.0

T2 seed selections of events generated by co-transformation of KS349 andKS362 were screened by GC analysis of seed chips as described above(Example 6). Seed were harvested from individual plants. Seed selectionsfrom heterozygous plants derived from transformations with Yarrowia DGATgenes segregated for elevated oleic acid content (>22% of total FA).Example 11 describes that event 4818.1.9 only contains an intactexpression cassette for the DGAT1 gene from Yarrowia. Oil content ofseed with elevated oleic acid content (with DGAT1 transgene) and nullsegregant seed from the same plant is shown in Table 17. In this table,oil content of seed containing the DGAT1 transgene was compared to thatof non-transgenic null segregant plants from the same plant.

TABLE 17 Oil content of transgenic seed and null segregant seed derivedfrom transgenic soybean T1 plants that segregate for a transgene withthe yarrowia DGAT1 gene average average oil (%) oil (%) Δ % Event PLANTw transg. n null n points Δ % 4818.1.9 A 21.8 58 18.9 42 4.4 25.1 B 21.316 17.0 6 C 23.1 34 17.0 14 average 22.1 17.6

Example 11 describes that other events generated by co-transformation ofKS349 and KS362 contain an intact expression cassette for both DGATgenes of Yarrowia. Oil content of seed with elevated oleic acid content(with both DGAT transgenes) and null segregant seed from the same plantis shown in Table 18. In this table, oil content of seed containing bothDGAT trangenes was compared to that of non-transgenic null segregantplants from the same plant.

TABLE 18 Oil content of transgenic seed and null segregant seed derivedfrom transgenic soybean T1 plants that segregate for transgenes withDGAT1 and DGAT2 genes from yarrowia average average oil (%) oil (%) Δ %Event PLANT w transg. n null n points Δ % 4818.1.2 A 23.9 33 20 15 3.417.3 B 22.9 35 20.3 13 C 23 31 19.2 17 average 23.3 19.8 4818.1.3 A 24.532 21.6 16 3.4 16.3 B 23.6 34 19.5 14 C 25.3 28 22 20 average 24.5 21.04818.2.6 A 22.3 31 19 17 3.2 15.8 B 24.1 21 20.6 27 C 24.1 23 20.7 25 D23.2 30 20.6 18 average 23.4 20.2T2 seed selection homozygous for the KS362 derived Yarrowia DGAT2expression cassette no longer segregated for the DS red marker. Oilcontent of 48 seed (or all available seed if less than 48 seed wereavailable) was measured by NMR. For each event DS-red negative T1 seedwere planted and T2 seed of null segregants were harvested from plantsgrown in the same growth chamber used for cultivation of T1 plantshomozygous for the DGAT transgene. Oil content of seed derived form nullsegregant selections and lines homozygous for the DGAT transgene inshown in Table 19.

TABLE 19 Oil content of null segregant seed and transgenic seed derivedfrom transgenic soybean T1 plants that are homozygous for transgeneswith yarrowia DGAT2 gene average Δ % EVENT PLANT n oil (%) points Δ %4822.1.13 A 48 22.7 2.3 11.6 B 48 21.9 C 48 22.7 average 22.4 NULL-A 4819.7 NULL-B 48 20.5 average 20.1 4822.1.9 A 48 23.0 3.6 18.5 B 48 24.0 C48 22.7 average 23.2 NULL-A 48 19.6 4822.2.10 A 48 22.5 2.3 10.9 B 4824.4 average 23.5 NULL-A 48 20.9 NULL-B 48 21.4 average 21.2T2 seed selection homozygous for KS349-derived Yarrowia DGAT1 andKS362-derived DGAT2 expression cassettes no longer segregated withrespect to the elevated oleic acid phenotype (≧22% oleic) associatedwith expression of yarrowia DGAT genes. Oil content of 48 seed (or allavailable seed if less than 48 seed were available) was measured by NMR.For each event T1 null segregant seed that showed no elevation oleicacid of were planted and T2 seed of these null segregants were harvestedfrom plants grown in the same growth chamber used for cultivation of T1plants homozygous for the DGAT transgenes. Oil content of seed derivedform null segregant selections and lines homozygous for the DGATtransgene in shown in Table 20.

TABLE 20 Oil content of null segregant seed and transgenic seed derivedfrom transgenic soybean T1 plants that are homozygous for transgeneswith DGAT1 and DGAT2 genes from yarrowia average Δ % EVENT PLANT n oil(%) points Δ % 4818.1.2 A 35 23.9 3.8 18.5 B 48 24.1 average 24.0 NULL-A48 20.1 NULL-B 48 20.4 average 20.3 4818.1.3 A 48 24.4 4.5 22.8 B 4824.4 C 48 24.3 average 24.4 NULL-A 48 20.2 NULL-B 48 19.5 average 19.94818.1.5 A 48 24.4 4.8 24.2 B 13 24.4 C 48 25.1 average 24.6 NULL-A 4819.4 NULL-B 48 20.2 average 19.8 4818.2.6 A 46 24.2 3.1 14.4 NULL-A 1121.4 NULL-B 48 20.9 average 21.2In summary, growth chamber results show excellent heritability of theincreased oil trait associated with overexpression of either a singleyarrowia DGAT genes or co-expression of both yarrowia DGAT genes insoybean seed. Oil increase (compared to null segregant seed) associatedwith expression of a single yarrowia DGAT genes is at least 10.9% and ashigh as 25.1%. Oil increase (compared to null segregant seed) associatedwith expression of both yarrowia DGAT genes is at least 14.4% and ashigh as 24.2%.

Example 13A Analysis of Transgenic Events: Field

The present example describes measurements of oil content of soybeanderived from T2 plants that were homozygous or heterozygous fortransgenes comprising YL DGAT1 or YL DGAT2 or both YL DGAT genes. T2plants were grown in a non-controlled environment (field).

DS red positive T1 seed of transgenic events generated with YL DGAT2(contained in KS 362) and corresponding DS red negative null segregantseed were grown in a field in Iowa in the summer of 2007. T1 seed withelevated oleic acid content that had been generated by co-transformationwith YL DGAT1 and YL DGAT2 and corresponding null segregant seed withnormal levels of oleic acid were grown in a similar fashion. T2 seedwere harvested from individual plants and subjected to NMR analysis tomeasure oil content. Table 21 shows oil content of 48 uniformly DS redpositive seed derived from events that were homozygous for the KS362transgene and that of DS-red negative seed from null segregant seed ofthe same event grown in the same environment.

TABLE 21 Oil content null segregant seed and transgenic seed derivedfrom field-grown transgenic soybean T1 plants that are homozygous fortransgenes with the yarrowia DGAT2 gene average Δ % EVENT PLANT n oil(%) points Δ % 4822.1.2 A 48 22.4 2.4 13 B 48 20.5 C 48 21.5 E 48 21.7 F48 20.7 G 40 19.5 H 16 23 average 21.3 Null A 48 18.9 Null B 48 19 NullC 48 18.7 Null D 16 17.9 Null E 24 20.2 average 18.9 4822.2.11 A 48 22.12.6 14 B 47 21 C 48 23.8 D 48 21.1 E 48 23.1 F 48 22 G 48 20.4 H 48 20.8I 48 20.3 average 21.6 Null A 48 19.2 Null B 48 19.5 Null C 48 18.3average 19.0 4822.2.10 A 40 21.8 2.6 13.6 B 48 21.8 average 21.8 Null A48 18.6 Null B 48 19.2 Null C 48 19.2 Null E 16 19.7 Null F 48 19.7 NullG 48 19 average 19.2Table 22 shows oil content of 48 seed from segregants that werehomozygous for YL DGAT1 and YL DGAT2 transgenes. All seed harvested fromthese homozygous T2 seed selections showed the elevated oleic acidcontent associated with YL DGAT expression. In Table 22 oil content ofthese lines is compared to that of null segregant seed of the sameevents with unaltered levels of oleic acid, derived from plants grown inthe same environment.

TABLE 22 Oil content null segregant seed and transgenic seed derivedfrom field-grown transgenic soybean T1 plants that are homozygous fortransgenes with the yarrowia DGAT1 and DGAT2 genes. average Δ % EVENTPLANT n oil (%) points Δ % 4818.1.2 A 37 22.2 3.8 20 B 48 22.5 C 48 22.7D 48 22.6 E 48 24.3 F 40 21.5 average 22.6 Null A 48 17.9 Null B 48 18.9Null C 48 19.4 Null D 48 19.5 Null E 48 19.7 Null F 48 18.4 Null G 4817.9 average 18.8 4818.1.3 A 48 23.5 3.5 19 B 48 22.4 C 48 22.8 D 1921.3 E 48 20.5 F 48 22.3 G 48 22.8 H 48 22.5 average 22.3 Null A 48 18.4Null B 48 19.1 Null C 48 17.9 Null D 48 19 Null E 48 19.3 Null F 48 18.3Null G 48 18.3 average 18.6

Example 11 describes that event 4818.1.9 only contains an intactexpression cassette for the DGAT1 gene from Yarrowia. Three T1 plants ofthis event derived from T1 seed with elevated oleic acid content weregrown in the Iowa field along side null segregant plants derived from T1seed with unaltered oleic acid content. T2 seed from all threetransgenic segregants still showed segregation of the elevated oleicacid phenotype indicating that the parental lines were stillheterozygous for the DGAT1 transgene. Using GC analysis alltransgene-positive seed were identified from these lines and subjectedto oil analysis by NMR. In Table 23 oil content of these seed iscompared to that of null segregant seed derived from T1 plants gown inthe same environment.

TABLE 23 Oil content null segregant seed and transgenic seed derivedfrom field-grown transgenic soybean T1 plants that are heterozygous fortransgenes with the yarrowia DGAT1 gene. average Δ % EVENT PLANT n oil(%) points Δ % 4818.1.9 A 14 20.7 2.0 11 B 14 19.1 C 20 20 average 20.0Null A 40 18.1 Null B 40 17.8 average 18.0

In summary, field environment results show excellent heritability of theincreased oil trait associated with overexpression of either a singleyarrowia DGAT genes or co-expression of both yarrowia DGAT genes insoybean seed. Oil increase (compared to null segregant seed) associatedwith expression of a single yarrowia DGAT genes is at least 11% and ashigh as 14%. Oil increase (compared to null segregant seed) associatedwith expression of both yarrowia DGAT genes is at least 19% and as highas 20%.

Example 13B Compositional Analysis of Soybean Seed

The present example describes measurements of seed compositionalparameters such as protein content and content of soluble carbohydratesof soybean seed derived from transgenic events that express single YLDGAT genes (YL DGAT2) of both YL DGAT genes.

Changes in the composition of soybean seed associated with expression ofYL DGAT genes were measured. To this end the concentrations of protein,soluble carbohydrates and starch were measured as follows.

Non-Structural Carbohydrate and Protein Analysis.

Dry soybean seed were ground to a fine powder in a GenoGrinder andsubsamples were weighed (to an accuracy of 0.1 mg) into 13×100 mm glasstubes; the tubes had Teflon® lined screw-cap closures. Three replicateswere prepared for each sample tested. Tissue dry weights were calculatedby weighing sub-samples before and after drying in a forced air oven for18 h at 105 C.

Lipid extraction was performed by adding 2 ml aliquots of heptane toeach tube. The tubes were vortex mixed and placed into an ultrasonicbath (VWR Scientific Model 750D) filled with water heated to 60 C. Thesamples were sonicated at full-power (˜360W) for 15 min and were thencentrifuged (5 min×1700 g). The supernatants were transferred to clean13×100 mm glass tubes and the pellets were extracted 2 more times withheptane (2 ml, second extraction, 1 ml third extraction) with thesupernatants from each extraction being pooled. After lipid extraction 1ml acetone was added to the pellets and after vortex mixing, to fullydisperse the material, they were taken to dryness in a Speedvac.

Non-Structural Carbohydrate Extraction and Analysis.

Two ml of 80% ethanol was added to the dried pellets from above. Thesamples were thoroughly vortex mixed until the plant material was fullydispersed in the solvent prior to sonication at 60 C for 15 min. Aftercentrifugation, 5 min×1700 g, the supernatants were decanted into clean13×100 mm glass tubes. Two more extractions with 80% ethanol wereperformed and the supernatants from each were pooled. The extractedpellets were suspended in acetone and dried (as above). An internalstandard 13-phenyl glucopyranoside (100 ul of a 0.5000+/−0.0010 g/100 mlstock) was added to each extract prior to drying in a Speedvac. Theextracts were maintained in a desiccator until further analysis.

The acetone dried powders from above were suspended in 0.9 ml MOPS(3-N[Morpholino]propane-sulfonic acid; 50 mM, 5 mM CaCl₂, pH 7.0) buffercontaining 100 U of heat stable α-amylase (from Bacillus licheniformis;Sigma A-4551). Samples were placed in a heat block (90 C) for 75 min andwere vortex mixed every 15 min. Samples were then allowed to cool toroom temperature and 0.6 ml acetate buffer (285 mM, pH 4.5) containing5U amyloglucosidase (Roche 110 202 367 001) was added to each. Sampleswere incubated for 15-18 h at 55 C in a water bath fitted with areciprocating shaker; standards of soluble potato starch (Sigma S-2630)were included to ensure that starch digestion went to completion.

Post-digestion the released carbohydrates were extracted prior toanalysis. Absolute ethanol (6 ml) was added to each tube and aftervortex mixing the samples were sonicated for 15 min at 60 C. Sampleswere centrifuged (5 min×1700 g) and the supernatants were decanted intoclean 13×100 mm glass tubes. The pellets were extracted 2 more timeswith 3 ml of 80% ethanol and the resulting supernatants were pooled.Internal standard (100 ul β-phenyl glucopyranoside, as above) was addedto each sample prior to drying in a Speedvac.

Sample Preparation and Analysis

The dried samples from the soluble and starch extractions describedabove were solubilized in anhydrous pyridine (Sigma-Aldrich P57506)containing 30 mg/ml of hydroxylamine HCl (Sigma-Aldrich 159417). Sampleswere placed on an orbital shaker (300 rpm) overnight and were thenheated for 1 hr (75 C) with vigorous vortex mixing applied every 15 min.After cooling to room temperature 1 ml hexamethyldisilazane(Sigma-Aldrich H-4875) and 100 ultrifluoroacetic acid (Sigma-AldrichT-6508) were added. The samples were vortex mixed and the precipitateswere allowed to settle prior to transferring the supernatants to GCsample vials.

Samples were analyzed on an Agilent 6890 gas chromatograph fitted with aDB-17MS capillary column (15 m×0.32 mm×0.25 um film). Inlet and detectortemperatures were both 275 C. After injection (2 ul, 20:1 split) theinitial column temperature (150 C) was increased to 180 C at a rate 3C/min and then at 25 C/min to a final temperature of 320 C. The finaltemperature was maintained for 10 min. The carrier gas was H₂ at alinear velocity of 51 cm/sec. Detection was by flame ionization. Dataanalysis was performed using Agilent ChemStation software. Each sugarwas quantified relative to the internal standard and detector responseswere applied for each individual carbohydrate (calculated from standardsrun with each set of samples). Final carbohydrate concentrations wereexpressed on a tissue dry weight basis.

Protein Analysis

Protein contents were estimated by combustion analysis on a ThermoFinnigan Flash 1112EA combustion analyzer. Samples, 4-8 mg, weighed toan accuracy of 0.001 mg on a Mettler-Toledo MX5 micro balance were usedfor analysis. Protein contents were calculated by multiplying % N,determined by the analyzer, by 6.25. Final protein contents wereexpressed on a % tissue dry weight basis.

TABLE 24 Compositional analysis of soybean seed derived from two T1plants that were either a null segregant or homozygous for an YL DGAT1YL DGAT2 transgene. The plants were grown in the same growth chamberenvironment. If not indicated otherwise values are reported as g/kg DW.Pini- Sorbi- Fruc- Glu- Myo- Su- Raffi- Stach- Total Protein Event toltol tose cose Inositol crose nose yose g/kg Starch (% DW) 4818.1.5 Mean1.57 0.26 4.60 1.61 0.27 47.0 8.01 35.88 99.2 0.45 38.6 NULL SD 0.030.02 0.18 0.27 0.01 1.6 0.15 0.72 2.0 0.03 0.3 4818.1.5 Mean 1.31 0.242.67 0.79 0.31 21.2 5.08 32.60 64.2 0.03 43.4 TG SD 0.09 0.00 0.07 0.020.01 0.4 0.11 1.21 1.7 0.02 0.2

TABLE 25 Compositional analysis of soybean seed derived from a T1 plantthat was heterozygous for an YL DGAT2 transgene. The plant was grown ina growth chamber. YL DGAT transgenic seed and null segregant seed wereselected based on DS red expression as a visible marker. Eight DS redpositive and DS red-negative seed were combined and analyzed asdescribed above. If not indicated otherwise values are reported as g/kgDW. Pini- Sorbi- Fruc- Glu- Myo- Su- Raffi- Stach- Total Protein Eventtol tol tose cose Inositol crose nose yose g/kg Starch (% DW) 4822.2.10Mean 1.66 0.17 5.50 2.57 0.28 47.2 6.64 45.0 108.9 0.31 40.2 NULL SD0.12 0.04 1.76 0.87 0.09 0.2 0.68 1.6 3.1 0.05 0.7 4822.2.10 Mean 2.020.19 5.02 1.72 0.33 32.7 6.91 41.4 90.3 0.31 40.8 TG SD 0.06 0.01 0.330.18 0.02 1.0 0.18 0.6 1.5 0.11 0.9

TABLE 26 Compositional analysis of soybean seed derived from T1 plantsthat were heterozygous for a YL DGAT2 or a YL DGAT1 YL DGAT2 transgene.The plants were grown in the field. YL DGAT transgenic seed and nullsegregant seed were selected based on DS red expression (4822.2.10) as avisible marker or elevated oleic acid content determined by GC analysis(4818.1.2). Eight transgene-positive and transgene-negative seed werecombined and analyzed as described above. If not indicated otherwisevalues are reported as g/kg DW. Pini- Sorbi- Fruc- Glu- Myo- Su- Raffi-Stach- Total Protein Event tol tol tose cose Inositol crose nose yoseg/kg Starch (% DW) 4822.2.10 Mean 2.44 0.26 1.16 1.12 0.44 57.7 8.5 39.0110.6 3.20 41.5 NULL SD 0.05 0.01 0.05 0.02 0.00 0.7 0.4 0.5 1.3 0.550.5 4822.2.10 Mean 1.89 0.35 0.82 0.67 0.32 29.7 5.5 31.7 70.9 1.00 46.1TG SD 0.06 0.09 0.14 0.06 0.01 1.5 0.1 0.5 2.2 0.23 0.7 4818.1.2 Mean1.79 0.51 1.17 1.12 0.37 49.8 6.1 42.0 102.8 0.51 44.5 NULL SD 0.02 0.010.13 0.13 0.01 2.6 0.4 2.4 5.1 0.07 2.2 4818.1.2 Mean 1.87 0.32 0.710.52 0.40 35.7 6.0 37.7 83.2 0.35 46.4 TG SD 0.05 0.02 0.05 0.01 0.020.8 0.2 1.5 2.5 0.08 0.2

Tables 24-26 illustrate that expression one or two YL DGAT genes indifferent environments (growth chamber, field) is associated with aconsistent shift in seed composition that is characterized by areduction in soluble carbohydrates, namely a reduction in sucrose and toa smaller extent a reduction in stachyose. Most importantly there is noreduction in protein content observed when oil accumulation is increasedthrough expression of YL DGAT genes.

Example 14 Measurements of DGAT Activity in Developing Seed and SomaticEmbryos

The present example describes construction of soybean expression vectorscomprising Yarrowia DGAT2 alone or Yarrowia DGAT2 and DGAT1, expressionof these gene(s) in soybean seed or somatic embryos and DGAT enzymeactivity in these tissues.

Construction of pKR1234 comprising YL DGAT2

The NotI fragment of KS362 (SEQ ID NO:52), containing the YL DGAT2, wascloned into the NotI fragment of pKR72 (SEQ ID NO:26; Example 4) toproduce pKR1234 (SEQ ID NO:68).

Construction of pKR1236 comprising YL DGAT 1 and DGAT2

The glycinin Gy1 promoter was PCR amplified from pZBL119 (which isdescribed in PCT Publication No. WO 2004/071467 and the contents ofwhich are hereby incorporated by reference) using primers oSGly-2 (SEQID NO:69) and oSGly-3 (SEQ ID NO:70). The resulting PCR fragment wassubcloned into the intermediate cloning vector pCR-Script AMP SK(+)(Stratagene), according to the manufacturer's protocol, to produceplasmid pPSgly32 (SEQ ID NO:71).

The PstI/NotI fragment of plasmid pSGly32 (SEQ ID NO:71), containing theGy1 promoter, was cloned into the PstI/NotI fragment from plasmid pKR142(which is described in PCT Publication No. WO 2004/071467), containingthe leguminA2 3′ transcription termination region, an ampicillinresistance gene, and bacterial ori, to produce pKR264 (SEQ ID NO:72).Thus, vector pKR264 contains a NotI site flanked by the promoter for theglycinin Gy1 gene and the leguminA2 3′ transcription termination region(Gy1/Not//legA2 cassette).

The NcoI/XbaI fragment of KS349 (SEQ ID NO:48), containing YarrowiaDGAT1, was cloned into the NcoI/XbaI sites of pKR908, (BB1574; publishedin US20080095915), which contains NcoI/XbaI sites flanked by NotI sites,to produce pKR1212 (SEQ ID NO:73).

The NotI fragment of pKR1212 (SEQ ID NO:73), containing the YarrowiaDGAT1 gene, was cloned into the NotI site of pKR264 (SEQ ID NO:72) toproduce pKR1235 (SEQ ID NO:74).

The BsiWI fragment of pKR1235 (74), containing the Yarrowia DGAT1, genewas cloned into the BsiWI site of pKR1234 (SEQ ID NO:68) to producepKR1236 (SEQ ID NO:75).

DGAT Assays on Microsomal Extracts from Developing T2 Seed

Soybean embryogenic suspension cultures (cv. Jack) were transformed withKS362 as described herein (Example 6), comprising Yarrowia DGAT2, asdescribed herein and T1 seed from soy plants AFS4822.1.13.1 (seed called7GR11-58) and AFS4822.2.10.1 (seed called 7GR11-66) were planted andplants grown as described in Example 12. In both of these events seedoil concentration of transgene positive seed was found to be elevatedwhen compared to null segregant seed of the same event.

Similarly, soybean embryogenic suspension cultures (cv. Jack) wereco-transformed with KS362 (comprising YL DGAT2) and KS349 (comprising YLDGAT1) as described herein (Example 6). Transgene-positive T1 seed fromevent AFS4818.1.2.1 are represented by seed 7GR11-2. In this event, seedoil concentration of transgene positive seed was found to be elevatedwhen compared to null segregant seed of the same event (Example 6).Transgene-negative, null segregant T1 seed derived from AFS4818.1.2.1and AFS4818.1.3.1 are represented by 7GR11-7 and 7GR11-15. These seedwere planted and plants grown as described in Example 12.

Approximately 1 g of T2 seed were collected from selected plants 30 daysafter flowering (DAF) and were snap frozen in liquid nitrogen and storedat −80 C until ready to process. After grinding 1 g of frozen seedtissue in liquid nitrogen in a mortar and pestle, 3 mL of planthomogenization buffer (300 mM sucrose; 1 mM EDTA; 10 mM Tris.HCl, pH8.0; 1 mM DTT; 0.1% polyvinylpolypyrrolidine) was added and tissue wasfurther homogenized using a polytron homogenizer for 1 minute. Debriswas collected by vacuum filtration through 3 layers of cheese clothfollowed by filtration through 1 layer of mira cloth. The resultingfiltrate was centrifuged for 15 min. twice at 1,500×g and the resultingsupernatant was then centrifuged at 100,000×g for 60 min. The resultingpellet was responded in approximately 0.5 to 1 mL of microsome buffer(100 mM potassium phosphate, pH 7.2) by gentle pipetting followed byfurther resuspension in a 2 mL sized Teflon-coated Dounce homogenizer.Protein concentrations were determine using Bradford reagent(Sigma-Aldrich) and microsomes were snap frozen in liquid nitrogen andstored at −80 C until assayed.

DGAT assays were carried out for 5 min at 25° C. in plant assay buffer(500 mM Tricine, pH 7.8; 28 mM sodium chloride; 0.06% CHAPS), with 20 μM1-14 C-labeled oleoyl-coenzyme A (50 mCi/mmol, Perkin Elmer), 1.5 mMdioleoylglyceride (Sigma-Aldrich) and 20 ng of microsomal protein in atotal reaction volume of 100 μl. Each reaction was initiated by additionof the microsomal membranes to the remainder of the reaction components.Assays were terminated by the addition of 1 mL of hot isopropanol (75 C)and heating at 75 for 10 min. Assays were cooled to RT, 1.5 mL of hexanewas added and samples were mixed. Phases were separated by low speedcentrifugation after addition of 1.25 ml of 500 mM sodium sulfate andthe upper phase was transferred to another glass tube. The top phase wasthen dried under nitrogen gas. The lipid from each assay wasre-dissolved in 75 uL of hexane spiked with 1 uL of soybean oil. Lipidwas applied to a Partisil K6 Silica Gel 60 A TLC plate (Whatman, 250 umthickness, 20 cm×20 cm) and triglycerides were separated from otherlipids by development with 80:20:1 (v/v/v) hexane: diethylether:aceticacid. Triacylglycerol was visualized and marked by light staining iniodine vapor in a tank. The plate was removed from the iodine tank andafter the stain faded, the triacylglycerol was scraped, andradioactivity determined by liquid scintillation counting and expressedas dpm per min. Total activity was determined as the amount ofradiolabeled oleic acid incorporated into triacylglycerol per minute permg of protein using the following formula: ([dpm]/[2200000 dpm/uCi]/[50uCi/umol]/[5 min.]/[0.02 mg protein]×[1000 nmol/umol]). DGAT activitiesfor each of the samples described are shown in Table 27.

TABLE 27 DGAT activities for selected developing T2 seed T2 DevelopingT1 Seed Seed DGAT Oil Activity (nmol · Exp. Plasmid DGAT Event T1 plantT1 Seed Phenotype min−1 · mg−1) 4818 KS362 DGAT2 AFS4818.1.2AFS4818.1.2.1 7GR1 DGAT 2.8 KS349 DAGT1 1-2 4818 KS362 DGAT2 AFS4818.1.2AFS4818.1.2.1 7GR1 null 0.4 KS349 DAGT1 1-7 4818 KS362 DGAT2 AFS4818.1.3AFS4818.1.3.1 7GR1 null 0.4 KS349 DAGT1 1-15 4822 KS362 DGAT2AFS4822.1.13 AFS4822.1.13.1 7GR1 DGAT 2.4 1-58 4822 KS362 DGAT2AFS4822.2.10 AFS4822.2.10.1 7GR1 DGAT 1.6 1-66DGAT Assays on Microsomal Extracts from Soybean Somatic Embryos

Soybean embryogenic suspension cultures (cv. Jack) were transformed witheither pKR1234 (SEQ ID NO:68), comprising Yarrowia DGAT2 and havingexperiment number MSE2181, or with pKR1236 (SEQ ID NO:75), comprisingYarrowia DGAT2 and DGAT1 and having experiment number MSE2182. Eventswere selected and somatic embryos matured in SHaM as described inExample 5.

After 2 weeks of maturation in SHaM, approximately 1 g of tissue fromeach event was frozen in liquid nitrogen and tissue was ground with amortar and pestle as described for soybean developing seed. A smallamount of ground tissue (approximately 100 mg) was lyophilized overnightand the remaining tissue was stored at −80 C.

Oil concentrations were determined on approximately 10 mg of lyophilizedtissue from each event using the GC method and 17:0 internal standardexactly as described in Example 5 and results for oil concentrations andoleic acid content (% of total FAME) are shown in Table 28. Microsomalprotein preparations were made, protein concentrations determined andDGAT assays were carried out on selected events determined to have arange of oil concentrations exactly as previously described for seedtissue. Results for DGAT assays are also shown in Table 28.

TABLE 28 Esterified Fatty Acid, Oleic Acid Content and DGAT activitiesof Soybean Somatic Embryos Transformed with either pKR1234 or pKR1236oleic acid Event FAME (% total DGAT Activity # (% DCW) FAME) (nmol ·min⁻¹ · mg⁻¹) MSE2181-pKR1234 (YL DGAT2) 8 8.9 31.9 3.6 5 7.3 33.8 4.5 36.6 32.6 2.5 9 6.2 30.7 2.5 7 6.1 30.4 16 5.8 25.4 1.6 12 5.6 26.1 155.4 26.7 2.8 4 4.6 22.5 0.7 11 4.2 22.7 0.5 14 4.0 21.2 13 3.8 23.2 1.86 3.5 25.3 10 3.2 21.2 0.5 MSE2182-pKR1236 (YL DGAT2/YL DGAT1) 2 10.938.8 6.3 7 10.8 38.8 4 10.3 38.4 5.1 3 10.0 38.0 8 9.8 38.1 4.4 17 9.629.3 10 8.6 33.3 5.7 28 8.2 36.3 13 8.1 43.6 5 8.0 25.4 20 8.0 36.4 127.7 29.3 4.8 26 7.6 30.5 24 7.2 33.4 9 6.3 20.4 0.7 29 6.3 29.8 21 6.131.1 19 5.7 32.5 6 5.6 21.7 0.5 23 5.6 33.5 16 5.4 26.3 25 5.1 20.3 145.1 26.2 0.9 1 5.0 24.9 18 4.9 22.2 30 4.5 17.0 0.4 27 3.7 19.7 15 3.221.6 11 2.9 16.6 2.5 22 2.8 20.7

Events transformed with pKR1234 and having some of the highest oilconcentrations had increases in DGAT activity of up to 9-fold comparedwith those events having wild-type levels of oil. Events transformedwith pKR1236 and having some of the highest oil concentrations hadincreases in DGAT activity of up to 15.8-fold compared with those eventshaving wild-type levels of oil.

Soybean embryogenic suspension culture (cv. Jack) was also transformedwith KS364 (SEQ ID NO:63), comprising Yarrowia DGAT2 and DGAT1(Experiment # MSE2134) and individual events were analyzed for fattyacid profile and oil concentration as described in Example 5. Based onthis data, one event (Event 54) having high oleic acid (32.83% of totalfatty acids) and oil concentrations (12.5% DCWt) and one event (Event33) having wild-type levels of oleic acid (15.52% of total fatty acids)and oil concentrations (8.2% DCWt) were chosen for DGAT assays.Transformed embryogenic suspension culture from each event was bulked upin SB 196 media and embryos matured in SHaM as described in Example 5.

After 2 weeks of maturation in SHaM, approximately 1 g of tissue fromeach event was frozen in liquid nitrogen, microsomal proteinpreparations were made and DGAT assays were carried out on each eventexactly as previously described and results are shown in Table 29. Totallipid was also extracted and oleic acid and oil concentrations weredetermined as described below and results are reported in Table 29.

TABLE 29 Esterified Fatty Acid, Oleic Acid Content and DGAT activitiesof Soybean Somatic Embryos Transformed with KS364 KS364 (DGAT2/DGAT1)oleic acid FAME (% (% total DGAT Activity Event # DCW) FAME) (nmol ·min⁻¹ · mg⁻¹)) 33 8.2 15.5 0.3 54 12.7 32.1 3.2

The event having the high oil concentration (Event 54) had increases inDGAT activity of 10.7-fold compared with the event having wild-typelevels of oil (Event 33).

Example 15 Analysis of Lipid Fractions of Transgenic Seed and SomaticEmbryos Expressing DGAT Genes

Soy somatic embryos transformed with KS364 from an event with wild-typeconcentrations of oil and oleic acid (Event 33) and from an event withhigh concentrations of oil and oleic acid (Event 54) were lyophilizedfor 48 hr and tissue was ground using a genogrinder exactly as describedin Example 5.

Total Lipid Extraction

Total lipid was extracted from each event by the method of Bligh, E. G.& Dyer, W. J. (Can. J. Biochem. Physiol. 37:911-917 (1959)) with somemodifications. Briefly, approximately 100 mg of ground tissue from eachevent was added to a 16 mm×125 mm sized test-tube with a teflon-linedscrew cap lid. A mixture of methanol:chloroform/2:1 (6 mL) was added andthe sample was extracted with gentle mixing for 1 hr after which 2 mL ofchloroform was added followed by continued mixing for 30 min.Afterwards, 3.6 mL of water was added, the tube was vortexed vigorouslyand phases were separated by centrifugation in a clinical centrifuge.The lower organic layer was gently removed to a second glass test tubeand the upper aqueous layers were re-extracted with 2 mL of chloroform.Centrifugation was repeated and the lower organic phase was combinedwith the first organic phase. Samples were dried under a stream ofnitrogen at 50 C, total lipid was estimated by weighing and lipid wasdissolved in chloroform:methanol/6:1 to a concentration of approximately10 mg/mL. FAME analysis was carried out on approximately 50 ug of eachsample using the sulfuric acid/methanol procedure described herein(Example 4) and results are shown in Table 30.

Separation of Neutral and Polar Lipids

Sep-pak amino-propyl solid phase extraction columns (Waters; 6 cccolumns, WAT054560) were equilibrated with 5 mL of methanol followed by5 mL of methanol:chloroform/1:1 followed by 5 mL of chloroform.Approximately 5 mg of total lipid in chloroform:methanol/6:1 was addedto each column, followed by 5×1 mL aliquots of chloroform to eluteneutral lipids and all fractions were collected, combined and driedunder a stream of nitrogen at 50 C. Polar lipids were then eluted fromeach column using 5×1 mL aliquots of methanol:chloroform/1:1 followed by5×1 mL aliquots of methanol and all fractions were combined and driedunder nitrogen. Neutral lipids were dissolved in approximately 1 mL ofCHCl₃:MeOH/6:1 and polar lipids were dissolved in approximately 200 uLof CHCl₃:MeOH/6:1. FAME analysis was carried out on approximately 50 ugof neutral lipid using the sulfuric acid/methanol procedure describedherein (Example 4) and results are shown in Table 30.

Separation of TAG, PC and PE by TLC

Approximately 100 uL of neutral lipid extract was loaded 2 cm from thebottom of a Partisil K6 Silica Gel 60 A TLC plate (Whatman, 250 umthickness, 20 cm×20 cm). Similarly, approximately 200 uL of the polarlipid fraction was loaded onto the same TLC plate. Standard solutions(10 mg/mL in chloroform:methanol/6:1) of TAG, PC and PE were alsospotted onto the plates. TLC plates were developed inCHCl3:MeOH:AcOH/65:35:8 until solvent front was approximately half wayup the plate. TLC plates were then air dried for 10 min and developedfully in 70:30:1 (v/v/v) hexane:diethylether:acetic acid. Standards werevisualized by light staining with iodine vapour and corresponding bandsfor TAG, PC and PE were cut out of the TLC plate. Silica gel containingeach lipid species was derivatized directly with sulfuric acid/methanolas described herein (Example 4) and results are shown in Table 30.

Fatty acid Positional Analysis of TAG

Fatty acid profiles of the sn2 position of TAG were determined usingporcine pancreatic lipase to remove acyl groups from the sn1 and sn3position of TAG only, followed by transesterification of the resultingmonoacylglyceride (MAG) produced. Approximately 5 mg of neutral lipidextract was suspended in 2 mL of 1M Tris.HCl, pH 8.0 along with 0.2 mLof 2.2% calcium chloride and 0.5 mL of 0.05% Bile salts in a glass screwcap test tube. The lipid was incubated at 37 C for 5 min, 5 mg ofporcine pancreatic lipase was added directly and the suspension wasincubated with shaking at 37 C for 20 min. After incubation, thereaction was reaction was terminated with the addition of 1 mL ofethanol followed by 1 mL of 6 M HCl. After mixing, 2.5 mL of diethylether was added, phases were separated by centrifugation and the toporganic layer was removed carefully. The diethyl ether extraction wasrepeated and the top diethyl ether phase was combined with the first.After drying over anhydrous sodium sulfate, the diethyl ether wasevaporated under a stream of nitrogen at 50 C and the resulting lipidwas dissolved in 200 uL of chloroform:methanol/6:1. The lipid was loadedonto a Partisil K6 TLC plate along with triacylglyceride (TAG),diacylglyceride (DAG), monoacylglyceride (MAG) and free fatty acid (FFA)standards and the TLC plate was developed as described herein.Afterwards, standards were visualized with light iodine staining and theMAG band was cut and derivatized with methanol/sulfuric acid aspreviously described herein. Results for the fatty acid profile of FAMEfrom the MAG band, representing the fatty acid profile of the sn2position of TAG (i.e. the acyl group on C2 of glycerol), along with thecalculated sn1 and sn3 positions, is shown in Table 30. In Table 30, the% of total fatty acid for each fatty acid (i.e. 16:0, 18:0, 18:1, 18:2,18:3) at the sn1 and sn3 positions of TAG is calculated with thefollowing formula:=([TAGx]−[sn2x]/3)*3/2; where the x indicates thefatty acid of interest.

TABLE 30 Fatty acid composition of various lipid species and positionaldistribution in TAG Sample Event % Oil 16:0 18:0 18:1 18:2 18:3 TotalExtract 33 8.2 15.6 3.9 15.5 50.8 14.2 54 12.7 10.5 4.7 32.1 43.9 8.8Neutral lipids 33 8.2 14.4 3.7 17.0 52.5 12.4 54 12.7 9.8 5.3 36.0 42.36.6 TAG 33 8.2 18.2 4.8 20.8 47.4 8.7 54 12.7 11.8 5.8 40.7 37.0 4.6 PC33 8.2 34.1 10.6 17.2 33.8 4.3 54 12.7 21.5 10.0 32.1 33.7 2.7 PE 33 8.241.1 6.8 13.4 25.9 12.8 54 12.7 45.8 9.8 21.9 15.3 7.1 TAG-sn1 33 8.21.0 0.3 14.9 72.7 11.0 54 12.7 1.0 0.5 24.0 67.1 7.4 TAG-sn1,3 33 8.226.8 7.0 23.8 34.9 7.5 (Calculated) 54 12.7 17.3 8.5 49.1 22.0 3.2Changes in fatty acid profiles associated with YL DGAT expressionobserved in TAG are also observed in polar lipids.

Example 16 Yarrowia DGAT Variants with Altered Amino Acid Sequence

The present example describes the creation of mutant forms of YarrowiaDGAT2, cloning them into a yeast expression vector and assayingmicrosomal protein fractions for DGAT activity.

Constructing Saccharomyces Expression Vectors Containing Mutant YarrowiaDGAT2s

Yarrowia DGAT2 was amplified from pKR1234 (SEQ ID NO:68; Example 14)with oligonucleotide primers oYDG2-1 (SEQ ID NO:76) and oYDG2-2 (SEQ IDNO:77), using the Phusion™ High-Fidelity DNA Polymerase (Cat. No. F553S,Finnzymes Oy, Finland) following the manufacturer's protocol. Theresulting DNA fragment was cloned into the pCR-Blunt® cloning vectorusing the Zero Blunt® PCR Cloning Kit (Invitrogen Corporation),following the manufacturer's protocol, to produce pKR1254 (SEQ IDNO:78).

A single codon in the Yarrowia DGAT2 sequence, which codes for aminoacid Y326 in the corresponding amino acid sequence SEQ ID NO:10, waschanged using the Quickchange® Site Directed Mutagenesis kit (Cat. No.200518, Stratagene, La Jolla, Calif.), with oligonucleotidesY1D2_Y326F-5 (SEQ ID NO:79) and Y1D2_Y326F-3 (SEQ ID NO:80), followingthe manufacturer's protocol. After extensive sequencing, a clone codingfor an amino acid sequence which is identical to the Yarrowia DGAT2 (SEQID NO:10), except that Y326 was changed to F326, was chosen for furtherstudy. This clone was designated pKR1254-Y326F (SEQ ID NO:81). Thenucleotide sequence for altered coding sequence (Y1DGAT2-Y326F) is setforth in SEQ ID NO:82 and the corresponding amino acid sequence is setforth in SEQ ID NO:83.

A single codon in the Yarrowia DGAT2 sequence, which codes for aminoacid Y326 in the corresponding amino acid sequence SEQ ID NO:10, waschanged using the Quickchange® Site Directed Mutagenesis kit (Cat. No.200518, Stratagene, La Jolla, Calif.), with oligonucleotidesY1D2_Y326L-5 (SEQ ID NO:84) and Y1D2_Y326L-3 (SEQ ID NO:85), followingthe manufacturer's protocol. After extensive sequencing, a clone codingfor an amino acid sequence which is identical to the Yarrowia DGAT2 (SEQID NO:10), except that Y326 was changed to L326, was chosen for furtherstudy. This clone was designated pKR1254_Y326L (SEQ ID NO:86). Thenucleotide sequence for altered coding sequence (Y1DGAT2_Y326L) is setforth in SEQ ID NO:87 and the corresponding amino acid sequence is setforth in SEQ ID NO:88.

A single codon in the Yarrowia DGAT2 sequence, which codes for aminoacid R327 in the corresponding amino acid sequence SEQ ID NO:10, waschanged using the Quickchange® Site Directed Mutagenesis kit (Cat. No.200518, Stratagene, La Jolla, Calif.), with oligonucleotidesY1D2_R327K-5 (SEQ ID NO:89) and Y1D2_R327K-3 (SEQ ID NO:90), followingthe manufacturer's protocol. After extensive sequencing, a clone codingfor an amino acid sequence which is identical to the Yarrowia DGAT2 (SEQID NO:10), except that R327 was changed to K327, was chosen for furtherstudy. This clone was designated pKR1254_R327K (SEQ ID NO:91). Thenucleotide sequence for altered coding sequence (Y1DGAT2_Y326L) is setforth in SEQ ID NO:92 and the corresponding amino acid sequence is setforth in SEQ ID NO:93.

The NotI fragments of pKR1254, pKR1254_Y326F, pKR1254_Y326L orpKR1254_R327K, each containing a wild-type or mutant version of YlDGAT2,were cloned into the NotI site of pY75 (SEQ ID NO:3; Example 1) toproduce pY191 (SEQ ID NO:94), pY192 (SEQ ID NO:95), pY193 (SEQ ID NO:96)or pY194 (SEQ ID NO:97), respectively.

Assaying DGAT activity of mutant Yarrowia DGAT2s

A mutant strain of Saccharomyces cerevisiae where the endogenous DGAT2gene (DGA1) was knocked out and has the following genotype (BY4741, MATahis3Δ1 leu2Δ0 met15Δ0 ura3Δ0) was obtained from Open Biosystems(http://www.openbiosystems.com/).

It was transformed with pY191, pY192, pY193 or pY194 and transformantswere isolated as described herein. Three individual transformants pertransformation were inoculated into 2 mL cultures of DOBA mediasupplemented with CSM-leu 30° C. for 16 h. Cells (1 mL) were transferredto 50 mL of DOBA medium described above and grown at 30° C. for anadditional 16 h. Cells were pelleted by centrifugation, frozen in liquidnitrogen and stored at −80° C. until required for use.

Pellets were re-suspended in 2 mL of yeast homogenization buffer (20 mMTris.HCl, pH 8.0; 10 mM MgCl2; 1 mM EDTA; 5% glycerol; 1 mM DTT; 0.3 M(NH₄)₂SO₄) and the suspension was added to a 2 mL screw cap tubecontaining approximately 1 mL of 0.5 mm glass beads. The after removalof air pockets by vortexing, the resuspension was filled to the top ofthe tube, the tube capped and the cells broken with three, 1 min. pulsesin a mini bead beater at 5000 rpm with storage on ice for 5 min. Theyeast homogenate was centrifuged at 1,500×g for 15 min at 4 C and theresulting supernatant was then centrifuged at 100,000×g for 60 min. Theresulting pellet was responded in approximately 0.2 to 0.5 mL ofmicrosome buffer (100 mM potassium phosphate, pH 7.2) by gentlepipetting followed by further resuspension in a 2 mL sized Teflon-coatedDounce homogenizer. Protein concentrations were determine using Bradfordreagent (Sigma-Aldrich) and microsomes were snap frozen in liquidnitrogen and stored at −80 C until assayed.

DGAT assays were carried out for 1 min at 25° C. in yeast assay buffer(50 mM potassium phosphate (pH 7.2)), with 20 μM 1-14C-labeledoleoyl-coenzyme A (50 mCi/mmol, Perkin Elmer), and 20 ng of microsomalprotein in a total reaction volume of 100 μl. Each reaction wasinitiated by addition of the microsomal membranes to the remainder ofthe reaction components. Assays were terminated and radioactivity intoTAG determined exactly as described for the plant DGAT assays except theformula was changed to reflect a 1 min. assay time (i.e. [dpm]/[2200000dpm/uCi]/[50 uCi/umol]/[5 min]/[0.02 mg protein]×[1000 nmol/umol]). DGATactivities for each of the samples as well as the averages described areshown in Table 31.

TABLE 31 DGAT activities for DGA1 Transformed with pY191, pY192, pY193or pY194. Avg. DGAT Activity DGAT Activity Std. Plasmid Mutant (nmol ·min^(−1·)mg⁻¹) (nmol · min^(−1·)mg⁻¹) Dev. pY191 wt 7.6 10.4 2.7 10.613.0 pY192 Y326F 6.5 8.3 1.7 8.4 10.0 pY193 Y326L 6.2 8.1 1.9 10.0 8.2pY194 R327K 5.9 6.2 0.4 6.0 6.7From Table 31, it appears that the Y326F and Y326L amino acid changeshave minimal effect on Yarrowia DGAT2 activity when assayed in yeast andthese two mutants were chosen for expression in soy somatic embryos.

Constructing Soy Expression Vectors Containing Mutant Yarrowia DGAT2s

The NotI fragment of pKR1254 (SEQ ID NO:78), pKR1254_Y326F (SEQ IDNO:81) or pKR1254_Y326L (SEQ ID NO:86), containing wild-type or mutantforms of Yarrowia DGAT2, were cloned into the NotI fragment of pKR72(SEQ ID NO:26; Example 4) to produce pKR1256 (SEQ ID NO:98), pKR1277(SEQ ID NO:99) or pKR1278 (SEQ ID NO:100), respectively.

Determining Oil Concentrations of Soy Somatic Embryos Expressing MutantYarrowia DGAT2s

Soybean embryogenic suspension culture (cv. Jack) was transformed witheither pKR1256 (SEQ ID NO:98), comprising wild-type Yarrowia DGAT2 andhaving experiment number MSE2228, pKR1277 (SEQ ID NO:99), comprisingYarrowia DGAT2_Y326F and having experiment number MSE2229, or pKR1278(SEQ ID NO:100), comprising Yarrowia DGAT2_Y326L and having experimentnumber MSE2230. Events were selected and somatic embryos matured in SHaMas described in Example 5. Oil concentrations were determined for eachevent using the NMR and described herein and fatty acid profiles weredetermined by GC exactly as described in herein and results for oilconcentrations and oleic acid content (% of total FAME) are shown inTable 32

TABLE 32 Oil concentrations for somatic soy embryos transformed withpKR1256, pKR1277 or pKR1278 MSE2228-pKR1256 MSE2229-pKR1277MSE2230-pKR1278 (wt DGAT2) (Y326F) (Y326L) oleic acid oleic acid oleicacid FAME (% total FAME (% total FAME (% total Event # (% DCW) FAME)Event # (% DCW) FAME) Event # (% DCW) FAME) 2228-9 14.3 44.4 2229-6 15.143.5 2230-4 11.6 35.1 2228-20 12.6 34.3 2229-20 14.4 44.3 2230-21 10.122.3 2228-3 12.4 39.3 2229-13 14.4 38.1 2230-15 9.8 22.4 2228-14 12.336.8 2229-21 14.0 36.5 2230-8 9.7 34.9 2228-2 11.7 36.8 2229-16 13.236.3 2230-29 9.6 33.0 2228-15 10.9 35.2 2229-24 13.1 39.9 2230-27 9.435.6 2228-5 10.1 23.3 2229-23 12.3 42.7 2230-3 9.4 36.7 2228-17 10.023.5 2229-3 12.2 41.3 2230-19 9.0 34.7 2228-24 10.0 27.6 2229-25 12.040.4 2230-6 8.6 23.6 2228-6 9.5 25.2 2229-31 11.9 39.0 2230-24 8.5 23.32228-18 9.5 32.4 2229-18 11.6 40.8 2230-18 8.4 35.8 2228-21 9.0 32.92229-5 11.4 38.7 2230-22 7.3 23.3 2228-12 8.7 22.7 2229-12 11.3 36.72230-2 7.0 26.7 2228-10 8.4 24.3 2229-10 11.3 35.6 2230-14 6.9 25.92228-22 7.5 29.9 2229-27 10.9 27.8 2230-9 6.9 22.9 2228-8 6.8 21.42229-30 10.7 39.0 2230-25 6.8 27.3 2228-19 6.5 27.3 2229-15 10.4 36.32230-7 6.8 37.5 2228-23 6.4 26.5 2229-8 9.7 39.5 2230-5 6.5 25.6 2228-76.1 22.2 2229-9 9.4 37.4 2230-10 6.4 31.1 2228-13 6.1 24.0 2229-22 9.222.3 2230-30 6.3 24.7 2228-11 5.2 24.3 2229-7 8.7 23.0 2230-26 6.3 23.42228-4 5.1 18.5 2229-26 8.4 31.2 2230-17 6.3 25.2 2228-16 4.2 19.32229-17 8.3 38.2 2230-20 6.3 19.5 2228-1 3.2 23.1 2229-28 7.9 27.22230-13 5.8 22.3 average 8.6 28.1 2229-29 7.8 25.7 2230-12 5.7 21.72229-1 7.4 32.8 2230-23 5.6 25.1 2229-2 5.9 18.6 2230-11 5.5 20.72229-11 4.6 18.6 2230-1 5.4 26.3 2229-19 4.1 22.6 2230-16 5.3 24.02229-4 4.0 21.2 2230-28 4.6 20.4 2229-14 3.1 39.8 2230-31 4.3 18.3average 8.8 32.2 average 6.5 25.4

In soy somatic embryos, a variant of the YL DGAT2 protein carrying theY326F mutation increases oil concentrations and shifts the fatty acidprofile of the oil at least to the same extent as the wild-type YarrowiaDGAT2.

Example 17 Expression Optimized DGAT Genes

Sequences encoding YL DGAT1 and YL DGAT2 genes that are optimized forexpression in soybean plants are set forth as SEQ ID NO:64 and SEQ IDNO:66. The design of these sequences is described in Example 9. DNAmolecules with this DNA sequence flanked by Not I restriction sites weresynthesized by DNA 2.0 (California, USA). Plasmid DNA with thesynthesized genes was digested with Not I. Not I restriction fragmentswith the DGAT genes were ligated to Not linearized, dephosphorylated DNAof KS332, which is described in Example 5. The resulting DNA constructsin which expression of expression-optimized variants of yarrowia DGAT1or yarrowia DGAT2 genes are under the control of the betaconglycininpromoter are henceforth referred to as KS392 and KS393. Their sequenceis set forth as SEQ ID NO:101 and SEQ ID NO:102. Moreover plasmid KS391was constructed. To this end DNA of KS349 was digested with NotI andNcoI. Ends of the of the resulting DGAT1 restriction fragment werecompletely filled-in and ligated to NotI linearized and filled in DNA ofKS332. The resulting plasmid construct is henceforth referred to asKS391. In this construct the native YL DGAT1 sequences is under thecontrol of the betaconglycinin promoter. The sequence of KS391 is setforth as SEQ ID NO:103. Transgenic soybean somatic embryos wereregenerated as after particle bombardment with plasmid DNA of KS391,KS392, KS 362 and KS393 as described above (Example 5). Oil content ofsomatic embryos was measured using NMR. Briefly lyophilized embryotissue was pulverized in genogrnder vial as described previously(Example 4). 20-200 mg of tissue powder were transferred to NMR tubes.Oil content of the somatic embryo tissue powder was calculated from theNMR signal as described in Example 4.

TABLE 33 Oil concentrations for somatic soy embryos transformed withpKS392 and pKS391 Construct KS392 Construct KS391 SAMPLE ID % oil SAMPLEID % oil 1 2196.1.08 15.7 1 2195.3.15 12.8 2 2196.3.03 13.9 2 2195.5.0211.5 3 2196.1.05 13.7 3 2195.3.01 11.2 4 2196.1.15 13.4 4 2195.3.03 11.05 2196.1.02 12.2 5 2195.3.02 10.6 6 2196.3.07 12.1 6 2195.4.06 10.4 72196.3.04 12.1 7 2195.3.08 10.2 8 2196.3.05 11.6 8 2195.2.01 10.1 92196.1.06 11.4 9 2195.3.04 9.3 10 2196.1.07 11.4 10 2195.3.05 9.2 112196.1.03 10.4 11 2195.5.01 9.1 12 2196.1.14 10.2 12 2195.2.04 8.8 132196.1.12 9.4 13 2195.3.14 8.6 14 2196.3.08 9.1 14 2195.3.10 7.8 152196.3.09 8.1 15 2195.4.01 7.7 16 2196.4.02 8.1 16 2195.5.03 6.5 172196.1.09 7.8 17 2195.4.02 6.4 18 2196.2.01 7.6 18 2195.3.09 6.3 192196.5.01 7.4 19 2195.4.07 6.2 20 2196.3.01 7.4 20 2195.3.06 6.1 212196.1.04 7.3 21 2195.3.13 6.0 22 2196.2.02 7.3 22 2195.5.05 5.9 232196.3.02 6.9 23 2195.4.03 5.6 24 2196.1.10 6.5 24 2195.5.04 5.5 252196.4.03 6.5 25 2196.1.01 5.3 26 2196.1.11 6.0 26 2195.4.04 5.3 272196.4.01 5.7 27 2195.3.11 5.2 28 2196.3.06 5.4 28 2195.2.03 5.0 292196.5.02 5.3 29 2195.4.05 4.9 30 2196.1.13 3.5 30 2195.3.07 4.4 312195.2.02 4.3 AVERAGE 9.1 AVERAGE 7.6 % OIL % OIL

Table 33 compares the oil content of 30 and 31 events generated withKS392 and KS 391, respectively. Average oil content of all eventsgenerated with KS392 was 9.1% whereas oil content of all eventsgenerated with KS391 was 7.6%. More over the highest oil contentobserved with KS392 was 15.7% compared to 12.8% for KS391. Applicantshave demonstrated that expression optimization of YL DGAT1 leads toincreased oil content in developing soybean embryos when compared to thenative YL DGAT 1 gene.

TABLE 34 Oil concentrations for somatic soy embryos transformed withpKS393 and pKS362 Construct KS393 Construct KS362 SAMPLE ID % oil SAMPLEID % oil 1 2207.5.05 12.3 1 2208.2.08 11.6 2 2207.5.08 12.2 2 2208.5.0411.5 3 2207.5.06 11.7 3 2208.2.04 11.5 4 2207.5.03 10.8 4 2208.2.10 11.25 2207.5.01 10.6 5 2208.2.09 10.3 6 2207.5.04 10.3 6 2208.2.02 10.2 72207.4.04 10.3 7 2208.5.02 10.0 8 2207.3.09 9.5 8 2208.3.10 9.8 92207.5.07 9.3 9 2208.3.12 9.8 10 2207.4.01 8.8 10 2208.3.06 9.5 112207.4.02 8.7 11 2208.3.04 8.8 12 2207.3.06 8.0 12 2208.5.05 8.6 132207.3.04 7.9 13 2208.2.07 8.1 14 2207.4.06 7.8 14 2208.2.03 8.0 152207.4.03 7.7 15 2208.3.03 8.0 16 2207.4.08 7.4 16 2208.5.01 8.0 172207.3.14 7.1 17 2208.2.06 7.8 18 2207.4.05 7.0 18 2208.2.11 7.4 192207.3.12 7.0 19 2208.5.07 7.3 20 2207.3.01 6.9 20 2208.3.14 7.2 212207.5.02 6.8 21 2208.3.02 7.0 22 2207.3.02 6.7 22 2208.3.08 6.4 232207.3.07 6.7 23 2208.3.07 6.1 24 2207.3.03 6.6 24 2208.2.12 6.1 252207.3.13 6.5 25 2208.3.09 6.0 26 2207.3.11 6.3 26 2208.3.11 6.0 272207.3.05 5.8 27 2208.2.01 5.8 28 2207.3.10 5.8 28 2208.2.05 5.7 292207.4.07 5.5 29 2208.3.01 5.4 30 2207.3.08 5.4 30 2208.5.03 5.1 312207.4.09 5.4 31 2208.3.13 4.7 32 2208.5.06 4.1 33 2208.3.05 3.2 AVERAGE8.0 AVERAGE 7.8 % OIL % OIL

Table 34 compares the oil content 31 of and 33 events generated withKS393 and KS 362. Average oil content of all events generated with KS393was 8.0% whereas oil content of all events generated with KS393 was7.8%. More over the highest oil content observed with KS393 was 12.3%compared to 11.6% for KS362. Applicants have demonstrated thatexpression optimization of YL DGAT2 leads a very small increase in oilcontent in developing soybean embryos when compared to the native YLDGAT2 gene.

Example 18 Cloning and Sequencing of DGAT2 from Torulospora delbrueckii

Isolation of Torulospora delbrueckii from a Cured Meat Sample

A Sopressata sausage was purchased from a local grocery store. A smallportion of the sausage, approximately 10 grams, was homogenized in asterile solution of 0.1% Triton-X 100 using a Waring blender. Glycerolwas added to the sausage suspension to a final concentration of 15%(w/w). Serial dilutions of the sausage suspension were plated on solidmedia prepared using premade Yeast Morphology Agar medium (HiMediaLaboratories, India) containing 150 mg L⁻¹ of chloramphenicol. Plateswere incubated at room temperature for one week. Individual colonieswere re-streaked on media with identical composition with the exceptionthat magenta-glucuro.CHA (Bromo-6-chloro-3-indolyl-β-D-glucuronidecyclohexylammonium salt (Sigma, USA)) was added to the medium at aconcentration of 200 mg L⁻¹.

Several yeast isolates did not produce dark-violet colonies whenstreaked on media containing magenta-glucuro.CHA. According to M. Quiroset al (Journal of Food Protection (2005), 68(4), 808-814) thiscolorimetric reaction provides reliable means for identification ofDebaryomyces hansenii.

Genomic DNA Isolation and Ribosomal DNA Sequencing

A liquid culture (50 mL YPD medium) of a magenta-glucuro.CHA-negativeyeast strain was grown at 28° C. for 72 h, at 250 rpm. Yeast cells werecollected by centrifugation and washed in deionized water. Yeast cellswere resuspended in 2 mL of STE buffer (0.1 M sorbitol, 10 mM Tris/HCl,pH 7.5, 1 mM EDTA) containing 10 mg mL⁻¹ of Zymolase (Zymo ResearchCorporation, California, USA) and incubated for 2 hours at 37° C. Totalgenomic DNA was isolated from Yeast spheroplasts as follows. The yeastsuspension was lysed in 4 mL of Urea Extraction buffer (0.3125 M NaCl,50 mM Tris HCl, pH 8.0, 20 mM EDTA, 1% sarkosine) and equal volume ofphenol/chloroform was added followed by thorough mixing. Aftercentrifugation the aqueous phase was re-extracted with phenolchloroform. Nucleic acids were precipitated by addition of ⅛ volume of4.4 M sodium acetate and 1 volume of isopropanol followed bycentrifugation. Nucleic acid were dried and resuspended in 500 microL TE(10 mM Tris, 1 mM EDTA). A small aliquot (1 microL) of the nucleic acidsolution was separated on 0.4% agarose gels next to molecular weightmarker to confirm that molecular weight was ≧50 kb.

TABLE 35 Oligonucleotide primers for amplification of a 26Sribosomal DNA sequence from ascomycete yeast name comment sequenceSEQ ID NO: MWG619 26s FWD GCATATCAATAAGCGGAGGAAAAG 104 MWG620 26s REVGGTCCGTGTTTCAAGACGG 105A 26S ribosomal gene fragment was generated by PCR as follows. The PCRreaction contained 50 mM KCl, 10 mM Tris-HCl (pH 9), 0.1% Triton X-100,2.5 mM MgCl₂, 0.2 mM each dNTP, 5 units of Taq polymerase (MBIFermentas, USA), 100 ng of genomic DNA of themagenta-glucuro.CHA-negative yeast strain and PCR primers MWG619 (SEQ IDNO:104) and MWG620 (SEQ ID NO:105) at a final concentration of 1 μM.Amplification was carried out for 35 cycles, each comprising 45 sec at94° C., 45 sec at 55° C., and 1 min at 72° C. A PCR product ofapproximately 600 bp was excised from agarose gels and sequenceddirectly using the primers MWG619 and MWG620. A BLASTN search against NT(Public DNA from NCBI) was conducted using the resulting DNA sequence.As shown in Table 36 the sequence was found to be 99% identical tovarious entries of 26s ribosomal sequences derived from Torulasporadelbrueckii. The 26s ribosomal DNA fragment of the Torulasporadelbrueckii yeast strain isolated by applicants from a cured meat sampleis set forth as SEQ ID NO:106.

TABLE 36 BLASTN results generated with the sequence of a magenta-glucuro.CHA-negative yeast strain derived from cured meat RankingSequence info Blast score 1 >gi|169125895|gb|EU441895.1| Score = 1082bits (546), Torulaspora delbrueckii strain Expect = 0.0 B-5(3) 26Sribosomal RNA Identities = 560/562 (99%), gene, partial sequence Gaps =2/562 (0%) Length = 613 Strand = Plus/Minus2 >gi|117573753|gb|EF063125.1| Score = 1082 bits (546), Torulasporadelbrueckii strain Expect = 0.0 EXOC35 26S ribosomal RNA Identities =560/562 (99%), gene, partial sequence Gaps = 2/562 (0%) Strand =Plus/Minus

Generation of DGAT2 Specific Probe Using PCR

A DGAT2 gene fragment was generated by PCR as follows. The PCR reactioncontained 50 mM KCl, 10 mM Tris-HCl (pH 9), 0.1% Triton X-100, 2.5 mMMgCl₂, 0.2 mM each dNTP, 5 units of Taq polymerase (MBI Fermentas, USA),100 ng of genomic DNA of Torulaspora delbrueckii and PCR primers P7 (SEQID NO:107) and P8 (SEQ ID NO:108) at a final concentration of 1 μM.Amplification was carried out for 35 cycles, each comprising 45 sec at94° C., 45 sec at 52° C., and 1 min at 72° C. A PCR product ofapproximately 270 bp was excised from agarose gels, cloned and sequencedusing standard techniques. One plasmid clones were identified thatcontained a cloned PCR product of 263 bp. The nucleotide and deducedamino acid sequence of this PCR product are set forth as SEQ ID NO:121and SEQ ID NO:122. A BLASTP search of the annotated fungal proteindatabase at NCBI was conducted. The deduced amino acid sequence of thePCR product shares 79.5% sequence identity to an unnamed protein productfrom Candida glabrata with GENBANK accession number XP 447864 whichrepresents the protein with the closest similarity in this database. Thededuced amino acid sequence of this PCR product also shares 75.9%sequence identity to Dga1 of Saccharomyces cerevisiae with GENBANKaccession number NP_(—)014888.

TABLE 37Degenerate oligonucleotide primers used for amplification of DGAT genes from oleaginousyeast and fungi gene oligonulceotide primer namedegenerate oligonucleotide sequence DGAT2 P7 (29 mer)AACTACATCTTCGGCTAYCAYCCNCAYGG (SEQ ID NO: 107) P8 (29 mer)AGGGACTCGGAGGCGCCGCCNCANACDAT (SEQ ID NO: 108) P7i (29 mer)AACTACATCTTCGGCTAYCAYCCXCAYGG (SEQ ID NO: 109) P8i (29 mer)AGGGACTCGGAGGCGCCGCCXCAXACDAT (SEQ ID NO: 110) BM DGAT2 fwd (41 mer)CCXCCXAAYMGXCCXTAYYTXTTYGGXTAYCAYCCXCAYGG (SEQ ID NO: 111)BM DGAT2 rev (40 mer)CRTTYTCXCCRAAXSWRAAXACXGGXACXARRTCXGCXCC (SEQ ID NO: 112) DGAT1fwd (37 mer) GGTGGGCXCCXACXYTXGTXTAYCARCCXGTXTAYCC (SEQ ID NO: 113)rev (41 mer) CCDATDATRTTRTGXGTXGGXACXCCXACXARXARYTCRTG (SEQ ID NO: 114)gene oligonulceotide primer name corresponding amino acid sequence DGAT2P7 (29 mer) NYIFGYHPHG (SEQ ID NO: 115) P8 (29 mer)IVVGGASESL (SEQ ID NO: 116) P7i (29 mer) NYIFGYHPHG (SEQ ID NO: 115)P8i (29 mer) IVVGGASESL (SEQ ID NO: 116) BM DGAT2 fwd (41 mer)PPNXPYXFGYHPH (SEQ ID NO: 117) BM DGAT2 rev (40 mer)GADXVPVFXFGEN (SEQ ID NO: 118) DGAT1 fwd (37 mer)WWAPTLVYQPVYP (SEQ ID NO: 119) rev (41 mer)HELLVGVPTHNIIG (SEQ ID NO: 120) Symbol (Meaning): R (G or A), Y (T orC), M (A or C), K (G or T), S (G or C), W (A or T), H (A or C or T), B(G or T or C), V (G or C or A), D (G or A or T), N (G or A or T or C),and X (Inosine)

Cosmid Library Construction, Screening and Sequencing of a DGAT2 Gene

Genomic DNA of Torulaspora delbrueckii was partially digested with MboI.Briefly, approximately 10 μg of genomic DNA were digested with 0.5 unitsof MboI (NEB, USA) in a final volume of 100 μL in the presence of 0.1 mgmL⁻¹ BSA, and 0.1 mg mL⁻¹ DNAse-free RNAse (Quiagen, USA) 100 mM NaCl,50 mM Tris-HCl, 10 mM MgCl₂, 1 mM DTT, pH 7.9. Aliquots of 25 μL wereremoved after 30, 60, 120 and 180 seconds respectively and combined in atube that contained 5 μL of 500 mM EDTA. The partially digested DNA waspurified and concentrated to a final volume of 10 μL using DNA Clean andConcentrator™ spin columns (Zymoresearch, USA) according to instructionsof the manufacturer. The DNA was ligated to 2 μg of linearized pLAFR3DNA (B. Staskawicz et al., Journal of bacteriology (1987), 169(12),5789-94) that had been completely digested with BamHI anddephosphorylated with Antarctic Phosphatase (NEB, USA). The ligationreaction was invitro-packaged and transfected into E. coli cells ofstrain NM554 (Stratagene, USA) using MAXPLAX packaging extracts(EPICENTRE Biotechnologies, USA) according to instructions of themanufacturer. Transfected cells were added to 15 mL of LB medium,incubated at 37° C. on Lab shaker set to 250 rpm. Glycerol was added toa final concentration of 15% (w/w) and the cell suspension was frozenusing a dry ice methanol mixture. The titer of the cosmid library wasdetermined by plating serial dilutions of the thawed cell suspension onsolidified LB medium containing 10 mg L⁻¹ of tetracycline. Approximately20,000 cosmid clones were plated on selective media, transferred toBiodyne B membranes (PALL Corporation, USA) and screened with a ³²Plabeled probe corresponding to the cloned PCR product derived fromTorulaspora delbrueckii (SEQ ID NO:121) according to stardard protocols.Cesium chloride density gradient centrifugation was used as described inT. Maniatis et al. Molecular Cloning: A Laboratory Manual. (1982), 545pp., was used to purify cosmid DNA from liquid cultures derived fromcolonies that hybridized to the probe under stringent conditions. Fivecosmid clones were sequenced using eight primers with SEQ ID NOs: 123through 130 (Table 38). Sequences were assembled using the SEQMANprogram of the LASERGENE™ 7.1.1 software package (DNASTAR, INC., USA).DNA sequencing of five independently isolated cosmids with said primersproduced DNA sequences that could be assembled into two closely relatedsequences. The consensus sequences derived from this effort are referredto as TD_DGAT2A gene and TD_DGAT2B gene and are set forth as SEQ IDNO:131 and SEQ ID NO:132. TD_DGAT2A gene and TD_DGAT2B gene are genomicsequences of 2700 bp that contain ORFs TD_DGAT2A (SEQ ID NO:133) andTD_DGAT2B (SEQ ID NO:134) that each comprise 1362 bp of sequenceencoding closely related proteins TD_DGAT2A (SEQ ID NO:135) andTD_DGAT2B (SEQ ID NO:136) that are both comprised of 453 amino acids.Nucleotide and deduced amino acid sequence of TD_DGAT2A and TD_DGAT2Bshare 95% and 96.9% sequence identity in CLUSTALW alignments. Thededuced amino acid sequence TD_DGAT2A and TD_DGAT2B share 57.4 and 56%sequence identity, respectively in a CLUSTALW alignment to an unnamedprotein product from Candida glabrata with GENBANK accession number XP447864 which represents the protein with the closest similarity to saidgenes from Torulaspora delbrueckii in this database. The deduced aminoacid sequences of TD_DAGT2A and TD_DGAT2B also share 57.3 and 57%sequence identity, respectively to Dga1 of Saccharomyces cerevisiae withGENBANK accession number NP_(—)014888.

TABLE 38 Primers used for sequencing of cosmid clonesderived from Torulaspora delbrueckii SEQ ID Name Sequence NO: MWG 625CCATTGGTACTGAAGGTTGTGGCTGGTCC 123 MWG 626 GGACCAGCCACAACCTTCAGTACCAATGG124 MWG 627 GTCCCATTGTACAGGGATTACTTATTGGCG 125 MWG 628CGCCAATAAGTAATCCCTGTACAATGGGAC 126 MWG 637 CCCTCATTATGGGCTTCCTAGGTTAG127 MWG 638 CTAACCTAGGAAGCCCATAATGAGGG 128 MWG 639CCAAACCCAACACAGGAACAAGTAGATC 129 MWG 640 GATCTACTTGTTCCTGTGTTGGGTTTGG130

Example 19 Cloning and Sequencing of DGAT2 from Pichia anomala

Isolation of Pichia anomala from a Cured Meat Sample

A Sopressata sausage was purchased from a local grocery store. A smallportion of the sausage, approximately 10 grams, was homogenized in asterile solution of 0.1% Triton-X 100 using a Waring blender. Glycerolwas added to the sausage suspension to a final concentration of 15%(w/w). Serial dilutions of the sausage suspension were plated on solidmedia prepared using premade Yeast Morphology Agar medium (HiMediaLaboratories, India) containing 150 mg L⁻¹ of chloramphenicol. Plateswere incubated at room temperature for one week. Individual colonieswere re-streaked on media with identical composition with the exceptionthat magenta-glucuro.CHA (Bromo-6-chloro-3-indolyl-β-D-glucuronidecyclohexylammonium salt (Sigma, USA)) was added to the medium at aconcentration of 200 mg L⁻¹.

Several yeast isolates did not produce dark-violet colonies whenstreaked on media containing magenta-glucuro.CHA. According to M. Quiroset al (Journal of Food Protection (2005), 68(4), 808-814) thiscolorimetric reaction provides reliable means for identification ofDebaryomyces hansenii.

Genomic DNA Isolation and Ribosomal DNA Sequencing

A liquid culture (50 mL YPD medium) of a secondmagenta-glucuro.CHA-negative yeast strain was grown at 28° C. for 72 h,at 250 rpm. Yeast cells were collected by centrifugation and washed indeionized water. Yeast cells were resuspended in 2 mL of STE buffer (0.1M sorbitol, 10 mM Tris/HCl, pH 7.5, 1 mM EDTA) containing 10 mg mL⁻¹ ofZymolase (Zymo Research Corporation, California, USA) and incubated for2 hours at 37° C. Total genomic DNA was isolated from Yeast spheroplastsas follows. The yeast suspension was lysed in 4 mL of Urea Extractionbuffer (0.3125 M NaCl, 50 mM Tris HCl, pH 8.0, 20 mM EDTA, 1% sarkosine)and equal volume of phenol/chloroform was added followed by thoroughmixing. After centrifugation the aqueous phase was re-extracted withphenol chloroform. Nucleic acids were precipitated by addition of ⅛volume of 4.4 M sodium acetate and 1 volume of isopropanol followed bycentrifugation. Nucleic acid were dried and resuspended in 500 microL TE(10 mM Tris, 1 mM EDTA). A small aliquot (1 microL) of the nucleic acidsolution was separated on 0.4% agarose gels next to molecular weightmarker to confirm that molecular weight was ≧50 kb.

A 26S ribosomal gene fragment was generated by PCR as follows. The PCRreaction contained 50 mM KCl, 10 mM Tris-HCl (pH 9), 0.1% Triton X-100,2.5 mM MgCl₂, 0.2 mM each dNTP, 5 units of Taq polymerase (MBIFermentas, USA), 100 ng of genomic DNA of the magenta-glucuro.CHA-negative yeast strain and PCR primers MWG619 (SEQ ID NO:104) andMWG620 (SEQ ID NO:105) at a final concentration of 1 μM. Amplificationwas carried out for 35 cycles, each comprising 45 sec at 94° C., 45 secat 55° C., and 1 min at 72° C. A PCR product of approximately 600 bp wasexcised from agarose gels and sequenced directly using the primersMWG619 (SEQ ID NO:104) and MWG620 (SEQ ID NO:105). A BLASTN searchagainst NT (Public DNA from NCBI) was conducted using the resulting DNAsequence. As shown in Table the sequence was found to be 99% identicalto various entries of 26s ribosomal sequences derived from Pichiaanomala. The 26s ribosomal DNA fragment of the Pichia anomala yeaststrain isolated by applicants from a cured meat sample is set forth asSEQ ID NO:137.

TABLE 39 BLASTN results generated with the sequence of a magenta-glucuro.CHA-negative yeast strain derived from cured meat RankingSequence info Sequence info 1 >gi|171194254|gb|EU560879.1| Score = 1031bits (520), Pichia anomala Expect = 0.0 strain 8 26S ribosomal RNA gene,Identities = 527/528 (99%), partial sequence Gaps = 1/528 Length = 604(0%) Strand = Plus/Minus 2 >gi|165967963|gb|EU327111.1| Score = 1031bits (520), Pichia anomala Expect = 0.0 strain TJY9d 26S ribosomalIdentities = 527/528 (99%), RNA gene, partial Gaps = 1/528 sequenceLength = 614 (0%) Strand = Plus/Minus

Generation of DGAT2 Specific Probe Using PCR

A DGAT2 gene fragment was generated by PCR as follows. The PCR reactioncontained 50 mM KCl, 10 mM Tris-HCl (pH 9), 0.1% Triton X-100, 2.5 mMMgCl₂, 0.2 mM each dNTP, 5 units of Taq polymerase (MBI Fermentas, USA),100 ng of genomic DNA of Pichia anomala and PCR primers P7 (SEQ IDNO:107) and P8 (SEQ ID NO:108) at a final concentration of 1 μM.Amplification was carried out for 35 cycles, each comprising 45 sec at94° C., 45 sec at 52° C., and 1 min at 72° C. A PCR product ofapproximately 270 bp was excised from agarose gels, cloned and sequencedusing standard techniques. One plasmid clones were identified thatcontained a cloned PCR product of 263 bp. The nucleotide and deducedamino acid sequence of this PCR product are set forth as SEQ ID NO:138and SEQ ID NO:139. A BLASTP search of the curated fungal proteindatabase at NCBI was conducted. Based on a CLUSTALW alignment, thededuced amino acid sequence of the PCR product shares 66.7% sequenceidentity to an unnamed protein product from Kluyveromyces lactis withGENBANK accession number XP_(—)455588 which represents the protein withthe closest similarity in this database. The deduced amino acid sequenceof this PCR product also shares 60.9% sequence identity to Dga1 ofSaccharomyces cerevisiae with GENBANK accession number NP_(—)014888.

Cosmid Library Construction, Screening and Sequencing of a DGAT2 Gene

Genomic DNA of Pichia anomala was partially digested with MboI. Briefly,approximately 10 μg of genomic DNA were digested with 0.5 units of MboI(NEB, USA) in a final volume of 100 μL in the presence of 0.1 mg mL⁻¹BSA, and 0.1 mg mL⁻¹ DNAse-free RNAse (Quiagen, USA) 100 mM NaCl, 50 mMTris-HCl, 10 mM MgCl₂, 1 mM DTT, pH 7.9. Aliquots of 25 μL were removedafter 30, 60, 120 and 180 seconds respectively and combined in a tubethat contained 5 μL of 500 mM EDTA. The partially digested DNA waspurified and concentrated to a final volume of 10 μL using DNA Clean andConcentrator™ spin columns (Zymoresearch, USA) according to instructionsof the manufacturer. The DNA was ligated to 2 μg of linearized pLAFR3DNA (B. Staskawicz et al., Journal of bacteriology (1987), 169(12),5789-94) that had been completely digested with BamHI anddephosphorylated with Antarctic Phosphatase (NEB, USA). The ligationreaction was invitro-packaged and transfected into E. coli cells ofstrain NM554 (Stratagene, USA) using MAXPLAX packaging extracts(EPICENTRE Biotechnologies, USA) according to instructions of themanufacturer. Transfected cells were added to 15 mL of LB medium,incubated at 37° C. on Lab shaker set to 250 rpm. Glycerol was added toa final concentration of 15% (w/w) and the cell suspension was frozenusing a dry ice methanol mixture. The titer of the cosmid library wasdetermined by plating serial dilutions of the thawed cell suspension onsolidified LB medium containing 10 mg L⁻¹ of tetracycline. Approximately20,000 cosmid clones were plated on selective media, transferred toBiodyne B membranes (PALL Corporation, USA) and screened with a ³²Plabeled probe corresponding to the cloned PCR product derived fromPichia anomala set forth as SEQ ID NO;138 according to stardardprotocols. Cesium chloride density gradient centrifugation was used asdescribed in T. Maniatis et al. Molecular Cloning: A Laboratory Manual.(1982), 545 pp., was used to purify cosmid DNA from liquid culturesderived from colonies that hybridized to the probe under stringentconditions. Five hybridization-positive cosmid clones were sequencedusing five primers with SEQ ID NOs 140 through 144 (Table 40). Sequenceswere assembled using the SEQMAN program of the LASERGENE™ 7.1.1 softwarepackage (DNASTAR, INC., USA). DNA sequencing of five independentlyisolated cosmids with said primers produced DNA sequences that could beassembled into a single sequence of 2062 bp referred to as PA_DAGT2 geneset forth as SEQ ID NO:145. It contains and ORF of 1593 bp referred toas PA_DGAT2 (SEQ ID NO:146) and encodes a protein with a deducedsequence of 429 amino acids set forth as SEQ ID NO:147. A BLASTP searchof the curated fungal protein database at NCBI was conducted. ThePA_DGAT2 gene product shares 48.2% sequence identity in a CLUSTALWalignment to a gene product derived GENBANK accession numberNP_(—)983542 from Ashbya gossypii ATCC 10895. This product is annotatedas having similarity to a protein family containing acyltransferasesinvolved in phospholipid biosynthesis and other proteins of unknownfunction. The deduced amino acid sequences of PA_DGAT2 also shares 46.1%sequence identity to Dga1 of Saccharomyces cerevisiae with GENBANKaccession number NP_(—)014888.

TABLE 40 Primers used for sequencing of cosmid clonesderived from Pichia anomala SEQ ID Name Sequence NO: MWG 653CCTTGAGTGGATTTGGTGGTATTGGAACTGACG 140 MWG 654CGTCAGTTCCAATACCACCAAATCCACTCAAGG 141 MWG 655GCCCTCAAAATATTGAAGCAAGGATTCTCC 142 MWG 656GGAGAATCCTTGCTTCAATATTTTGAGGGC 143 MWG 660 GGTGGTATTGGAACTGACGG 144

Example 20 Cloning and Sequencing of DGAT2 from Debaryomyces hansenii

Isolation of Debaryomyces Hansenii from a Cured Meat Sample

A Sopressata sausage was purchased from a local grocery store. A smallportion of the sausage, approximately 10 grams, was homogenized in asterile solution of 0.1% Triton-X 100 using a Waring blender. Glycerolwas added to the sausage suspension to a final concentration of 15%(w/w). Serial dilutions of the sausage suspension were plated on solidmedia prepared using premade Yeast Morphology Agar medium (HiMediaLaboratories, India) containing 150 mg L⁻¹ of chloramphenicol. Plateswere incubated at room temperature for one week. Individual colonieswere re-streaked on media with identical composition with the exceptionthat magenta-glucuro.CHA (Bromo-6-chloro-3-indolyl-β-D-glucuronidecyclohexylammonium salt (Sigma, USA)) was added to the medium at aconcentration of 200 mg L⁻¹.

Several yeast isolates produced dark-violet colonies when streaked onmedia containing magenta-glucuro.CHA. According to M. Quiros et al(Journal of Food Protection (2005), 68(4), 808-814) this colorimetricreaction provides reliable means for identification of Debaryomyceshansenii.

Genomic DNA Isolation

A liquid culture (50 mL YPD medium) of a magenta-glucuro.CHA-positveyeast strain was grown at 28° C. for 72 h, at 250 rpm. Yeast cells werecollected by centrifugation and washed in deionized water. Yeast cellswere resuspended in 2 mL of STE buffer (0.1 M sorbitol, 10 mM Tris/HCl,pH 7.5, 1 mM EDTA) containing 10 mg mL⁻¹ of Zymolase (Zymo ResearchCorporation, California, USA) and incubated for 2 hours at 37° C. Totalgenomic DNA was isolated from Yeast spheroplasts as follows. The yeastsuspension was lysed in 4 mL of Urea Extraction buffer (0.3125 M NaCl,50 mM Tris HCl, pH 8.0, 20 mM EDTA, 1% sarkosine) and equal volume ofphenol/chloroform was added followed by thorough mixing. Aftercentrifugation the aqueous phase was re-extracted with phenolchloroform. Nucleic acids were precipitated by addition of ⅛ volume of4.4 M sodium acetate and 1 volume of isopropanol followed bycentrifugation. Nucleic acid were dried and resuspended in 500 microL TE(10 mM Tris, 1 mM EDTA). A small aliquot (1 microL) of the nucleic acidsolution was separated on 0.4% agarose gels next to molecular weightmarker to confirm that molecular weight was ≧50 kb.

A DGAT2 gene fragment was generated by PCR as follows. The PCR reactioncontained 50 mM KCl, 10 mM Tris-HCl (pH 9), 0.1% Triton X-100, 2.5 mMMgCl₂, 0.2 mM each dNTP, 5 units of Taq polymerase (MBI Fermentas, USA),100 ng of genomic DNA of the magenta-glucuro.CHA-positve yeast strainand PCR primers P7 (SEQ ID NO:107) and P8 (SEQ ID NO:108) at a finalconcentration of 0.2 μM. Amplification was carried out for 35 cycles,each comprising 45 sec at 94° C., 45 sec at 52° C., and 1 min at 72° C.A PCR product of approximately 350 bp was excised from agarose gels,cloned and sequenced using standard techniques. Several plasmid cloneswere identified that contained a cloned PCR product of 328 bp. Thenucleotide and deduced amino acid sequence of this PCR product are setforth as SEQ ID NO:148 and SEQ ID NO:149. Based on a CLUTALW alignmentthe deduced amino acid sequence of the PCR product shares 95.4% sequenceidentity to hypothetical protein DEHAOC13101 g from Debaryomyceshansenii, CBS767. This gene which represents the best hit in publicsequence databases has GENBANK number XP_(—)458203 and is annotated ashaving similarity to acyltransferases involved in phospholipidbiosynthesis and other proteins of unknown function. Cosmid libraryconstruction, screening and sequencing of a DGAT2 gene Genomic DNA ofthe magenta-glucuro.CHA-positve yeast strain was partially digested withMboI. Briefly, approximately 10 μg of genomic DNA were digested with 0.5units of MboI (NEB, USA) in a final volume of 100 μL in the presence of0.1 mg mL⁻¹ BSA, and 0.1 mg mL⁻¹ DNAse-free RNAse (Quiagen, USA) 100 mMNaCl, 50 mM Tris-HCl, 10 mM MgCl₂, 1 mM DTT, pH 7.9. Aliquots of 25 μLwere removed after 30, 60, 120 and 180 seconds respectively and combinedin a tube that contained 5 μL of 500 mM EDTA. The partially digested DNAwas purified and concentrated to a final volume of 10 μL using DNA Cleanand Concentrator™ spin columns (Zymoresearch, USA) according toinstructions of the manufacturer. The DNA was ligated to 2 μg oflinearized pLAFR3DNA (B. Staskawicz et al., Journal of bacteriology(1987), 169(12), 5789-94) that had been completely digested with BamHIand dephosphorylated with Antarctic Phosphatase (NEB, USA). The ligationreaction was invitro-packaged and transfected into E. coli cells ofstrain NM554 (Stratagene, USA) using MAXPLAX packaging extracts(EPICENTRE Biotechnologies, USA) according to instructions of themanufacturer. Transfected cells were added to 15 mL of LB medium,incubated at 37° C. on Lab shaker set to 250 rpm. Glycerol was added toa final concentration of 15% (w/w) and the cell suspension was frozenusing a dry ice methanol mixture. The titer of the cosmid library wasdetermined by plating serial dilutions of the thawed cell suspension onsolidified LB medium containing 10 mg L⁻¹ of tetracycline. Approximately20,000 cosmid clones were plated on selective media, transferred toBiodyne B membranes (PALL Corporation, USA) and screened with a ³²Plabeled probe corresponding to the cloned PCR product derived from themagenta-glucuro.CHA-positve yeast strain according to standardprotocols. Cesium chloride density gradient centrifugation was used asdescribed in T. Maniatis et al. Molecular Cloning: A Laboratory Manual.(1982), 545 pp., was used to purify cosmid DNA from liquid culturesderived from colonies that hybridized to the probe under stringentconditions. Cosmid clones were sequenced using ten primers with SEQ IDNOs 150 through 159 (Table 41). Sequences were assembled using theSEQMAN program of the LASERGENE™ 7.1.1 software package (DNASTAR, INC.,USA). DNA sequencing of three independently isolated cosmids with saidprimers produced DNA sequences that could be assembled into a singlesequence. The consensus sequence of 2800 nucleotides derived from thiseffort is set for as SEQ ID NO:160. It contains an ORF of 2028 bp (SEQID NO:161) that can be translated into a protein of 675 amino acids (SEQID NO:162). This protein shares 91.3% sequence identity in a ClustalWalignment to hypothetical protein DEHAOC13101g from Debaryomyceshansenii, CBS767. This gene has GENBANK number XP_(—)458203. Itrepresents the gene with the closest similarity to the gene of SEQ IDNO:161 and is annotated as having similarity to acyltransferasesinvolved in phospholipid biosynthesis and other proteins of unknownfunction.

TABLE 41 Primers used for sequencing of cosmid clonesderived from Debaryomyces hansenii SEQ ID Sequence NO: MWG 621CGGAATGAACCATATCAGCCTCC 150 MWG 622 GGAGGCTGATATGGTTCATTCCG 151 MWG 623GAGCTTGGGATTGACAAGTGCATCTTAC 152 MWG 624 GTAAGATGCACTTGTCAATCCCAAGCTC153 MWG 631 GGCTGGATCATCTGGATATTTGTGATCC 154 MWG 632GGATCACAAATATCCAGATGATCCAGCC 155 MWG 633 GCCAGGATGATGACTCCTCAAGTCCAAG156 MWG 634 CTTGGACTTGAGGAGTCATCATCCTGGC 157 MWG 635GCTAACCCAAAACCGGGATCTCTTGG 158 MWG 636 CAGTGCCATTTTTCAGTGCCAGAGGTG 159

Example 21 Cloning and Sequencing of DGAT2 from Candida Zeylanoides

Isolation of Candida zeylanoides from a Cured Meat Sample

A sample of Parma Ham imported from Parma, Italy was purchased from alocal Italian deli store. A small portion of the ham, approximately 10grams, was homogenized in a sterile solution of 0.1% Triton-X 100 usinga Waring blender. Glycerol was added to the sausage suspension to afinal concentration of 15% (w/w). Serial dilutions of the sausagesuspension were plated on solid media prepared using premade YeastMorphology Agar medium (HiMedia Laboratories, India) containing 150 mgL⁻¹ of chloramphenicol. Plates were incubated at room temperature forone week. Individual colonies were re-streaked on media with identicalcomposition and used for strain identification by 26S ribosomal DNAsequencing. Briefly, a small protein of yeast cells were added to 100 μLof STE buffer containing 10 mg mL⁻¹ zymolase. Cells were resuspended andincubated for 30 min at 37° C. The cell suspension was heated to 95° C.for 10 min and cooled to room temperature. 5 μL of cell lysine were usedin a PCR reaction for amplification of a 26S ribosomal DNA fragment. ThePCR reaction contained 50 mM KCl, 10 mM Tris-HCl (pH 9), 0.1% TritonX-100, 2.5 mM MgCl₂, 0.2 mM each dNTP, 5 units of Taq polymerase (MBIFermentas, USA), 5 uL of yeast cell lysate and PCR primers MWG619 (SEQID NO:104) and MWG620 (SEQ ID NO:105) at a final concentration of 1 μM.Amplification was carried out for 35 cycles, each comprising 45 sec at94° C., 45 sec at 55° C., and 1 min at 72° C. PCR products ofapproximately 600 bp were excised from agarose gels and sequenceddirectly using the primers MWG619 (SEQ ID NO:104) and MWG620 (SEQ IDNO:105). A BLASTN search against NT (Public DNA from NCBI) was conductedusing the resulting DNA sequence. The vast majority of yeast coloniesderived from the Parma Ham samples produced PCR products that whensubjected to DNA sequencing showed >99% identity to 26S ribosomal DNAsequences of Debaryomyces hansenii. However, one yeast colony withmorphological features different from those of the majority of yeastcolonies derived from the Parma Ham sample could be identified thatproduced a 26S ribosomal genomic DNA sequence (SEQ ID NO:163) thatshowed high similarity (99% identity) to a sequence from Candidazeylanoides (Table)

TABLE 42 BLASTN results generated with 26s ribosomal sequence from ayeast strain derived from Parma Ham Ranking Sequence info Blast score1 >gi|165967959|gb|EU327107.1| Score = 1068 bits (539), Candidazeylanoides strain Expect = 0.0 TJY7b 26S ribosomal RNA gene, Identities= 549/551 (99%), partial sequence Length = 615 Gaps = 1/551 (0%) Strand= Plus/Plus 2 >gi|165967958|gb|EU327106.1| Score = 1068 bits (539),Candida zeylanoides strain Expect = 0.0 TJY18b 26S ribosomal RNA gene,Identities = 549/551 (99%), partial sequence Length = 605 Gaps = 1/551(0%) Strand = Plus/Plus Based on this result the strain was identifiedas Candida zeylanoides.

Genomic DNA Isolation

A liquid culture (50 mL, YPD medium) of the Candida zeylanoides strainwas grown at 28° C. for 72 h, at 250 rpm. Yeast cells were collected bycentrifugation and washed in deionized water. Yeast cells wereresuspended in 2 mL of STE buffer (0.1 M sorbitol, 10 mM Tris/HCl, pH7.5, 1 mM EDTA) containing 10 mg mL⁻¹ of Zymolase (Zymo ResearchCorporation, California, USA) and incubated for 2 hours at 37° C. Totalgenomic DNA was isolated from Yeast spheroplasts as follows. The yeastsuspension was lysed in 4 mL of Urea Extraction buffer (0.3125 M NaCl,50 mM Tris HCl, pH 8.0, 20 mM EDTA, 1% sarkosine) and equal volume ofphenol/chloroform was added followed by thorough mixing. Aftercentrifugation the aqueous phase was re-extracted with phenolchloroform. Nucleic acids were precipitated by addition of ⅛ volume of4.4 M sodium acetate and 1 volume of isopropanol followed bycentrifugation. Nucleic acid were dried and resuspended in 500 microL TE(10 mM Tris, 1 mM EDTA). A small aliquot (1 microL) of the nucleic acidsolution was separated on 0.4% agarose gels next to molecular weightmarker to confirm that molecular weight was ≧50 kb.

Generation of DGAT2 Specific Probe Using PCR

A DGAT2 gene fragment was generated by PCR as follows. The PCR reactioncontained 50 mM KCl, 10 mM Tris-HCl (pH 9), 0.1% Triton X-100, 2.5 mMMgCl₂, 0.2 mM each dNTP, 5 units of Taq polymerase (MBI Fermentas, USA),100 ng of genomic DNA of Candida zeylanoides and PCR primers P7 (SEQ IDNO:107) and P8 (SEQ ID NO:108) at a final concentration of 1 μM.Amplification was carried out for 35 cycles, each comprising 45 sec at94° C., 45 sec at 52° C., and 1 min at 72° C. A PCR product ofapproximately 330 bp was excised from agarose gels, cloned and sequencedusing standard techniques. One plasmid clones were identified thatcontained a cloned PCR product of 325 bp. The nucleotide and deducedamino acid sequence of this PCR product are set forth as SEQ ID NO:164and SEQ ID NO:165. A BLASTP search of the curated fungal proteindatabase at NCBI was conducted. Based on a CLUSTALW alignment, thededuced amino acid sequence of the PCR product shares 79.6% sequenceidentity to a protein product from Pichia stipidis with GENBANKaccession number XP_(—)001382973. This protein product is annotated ashypothetical protein with similarity to diacylglycerol acyltransferases.It represents the protein with the closest similarity in this database.

Cosmid Library Construction, Screening and Sequencing of a DGAT2 Gene

Genomic DNA of Candida zeylanoides was partially digested with MboI.Briefly, approximately 10 μg of genomic DNA were digested with 0.5 unitsof MboI (NEB, USA) in a final volume of 100 μL in the presence of 0.1 mgmL⁻¹ BSA, and 0.1 mg mL⁻¹ DNAse-free RNAse (Quiagen, USA) 100 mM NaCl,50 mM Tris-HCl, 10 mM MgCl₂, 1 mM DTT, pH 7.9. Aliquots of 25 μL wereremoved after 30, 60, 120 and 180 seconds respectively and combined in atube that contained 5 μL of 500 mM EDTA. The partially digested DNA waspurified and concentrated to a final volume of 10 μL using DNA Clean andConcentrator™ spin columns (Zymoresearch, USA) according to instructionsof the manufacturer. The DNA was ligated to 2 μg of linearized pLAFR3DNA(B. Staskawicz et al., Journal of bacteriology (1987), 169(12), 5789-94)that had been completely digested with BamHI and dephosphorylated withAntarctic Phosphatase (NEB, USA). The ligation reaction wasinvitro-packaged and transfected into E. coli cells of strain NM554(Stratagene, USA) using MAXPLAX packaging extracts (EPICENTREBiotechnologies, USA) according to instructions of the manufacturer.Transfected cells were added to 15 mL of LB medium, incubated at 37° C.on Lab shaker set to 250 rpm. Glycerol was added to a finalconcentration of 15% (w/w) and the cell suspension was frozen using adry ice methanol mixture. The titer of the cosmid library was determinedby plating serial dilutions of the thawed cell suspension on solidifiedLB medium containing 10 mg L⁻¹ of tetracycline. Approximately 20,000cosmid clones were plated on selective media, transferred to Biodyne Bmembranes (PALL Corporation, USA) and screened with a ³²P labeled probecorresponding to the cloned PCR product derived from Pichia anomalaaccording to standard protocols. Cesium chloride density gradientcentrifugation was used as described in T. Maniatis et al. MolecularCloning: A Laboratory Manual. (1982), 545 pp., was used to purify cosmidDNA from liquid cultures derived from colonies that hybridized to theprobe under stringent conditions. Four cosmid clones were sequencedusing eight primers with SEQ ID NOs 166 through 173 (Table). Sequenceswere assembled using the SEQMAN program of the LASERGENE™ 7.1.1 softwarepackage (DNASTAR, INC., USA). DNA sequencing of four independentlyisolated cosmids with said primers produced DNA sequences that could beassembled into a single sequence of 3021 bp referred to as CZ_DAGT2 geneset forth as SEQ ID NO:174. It contains and ORF of 1695 bp referred toas CZ_DGAT2 (SEQ ID NO:175) and encodes a protein with a deducedsequence of 564 amino acids set forth as SEQ ID NO:176. A BLASTP searchof the curated fungal protein database at NCBI was conducted. Based on aCLUSTALW alignment, the CZ_DGAT2 gene product shares 57.6% sequenceidentity in a CLUSTALW alignment to a gene product derived GENBANKaccession number XP_(—)001527478 from Lodderomyces elongisporus, NRRLYB-4239. This product is annotated as hypothetical protein withsimilarity to diacylglycerol acyltransferases. It represents the proteinwith the closest similarity in this database.

TABLE 43 Primers used for sequencing of cosmid clonesderived from Candida zeylanoides SEQ ID Name Sequence NO: MWG722GTCATTTCGATGGGGGTGATGGGCAC 166 MWG723 GTGCCCATCACCCCCATCGAAATGAC 167MWG724 GCGCCGCGGCAAAGAACATCAAGAGC 168 MWG725 GCTCTTGATGTTCTTTGCCGCGGCGC169 MWG726 CCCACTGGCGGGCGCCGACAGCACGC 170 MWG727GCGTGCTGTCGGCGCCCGCCAGTGGG 171 MWG728 GCGCGTCTACGAGGATAACAAGGACAAG 172MWG729 CTTGTCCTTGTTATCCTCGTAGACGCGC 173

Example 22 Cloning and Sequencing of DGAT 1 and DGAT2 from Lipomycesstarkeyi

RNA Isolation and cDNA Synthesis

Lipomyces starkeyi (Catalog number 78-23T) was purchased from the PHAFFcollection (UC Davis, Calif., USA). A liquid culture (50 mL, YPD medium)of Lipomyces starkeyi was grown at 28° C. Cells were harvested bycentrifugation washed with deonized water and collected again bycentrifugation. Total RNA was isolated from the resulting cell culturepellet using the hot phenol method exactly as described in CurrentProtocols in Molecular Biology (John Wiley & Sons, Somerset, N.J.) butwhere all reagent volumes were increased by 10-fold (e.g. cell pelletwas re-suspended in 4 mL of TES solution instead of 400 μL). The finalRNA pellet was dissolved in 1 mL of water and the concentration wasdetermined to be 500 ng/uL.

cDNA was synthesized from 1 μg of total RNA (2 μl) using theSuperScript™ First-Strand Synthesis System for RT-PCR Kit (Invitrogen™Life Technologies, Carlsbad, Calif.) with the provided oligo(dT) primeraccording to the manufacturer's protocol. After RNase H treatment thecDNA was used as PCR template for generation of DGAT1 And DGAT2-specificgene fragments as follows.

Generation of a DGAT1 Specific Probe Using PCR

A DGAT1 gene fragment was generated by PCR as follows. The PCR reactioncontained 50 mM KCl, 10 mM Tris-HCl (pH 9), 0.1% Triton X-100, 2.5 mMMgCl₂, 0.2 mM each dNTP, 5 units of Taq polymerase (MBI Fermentas, USA),2 μL of Lipomyces starkeyi cDNA and PCR primers DGAT1 FWD (SEQ IDNO:113) and DGAT1 REV (SEQ ID NO:114) at a final concentration of 1 μM.Amplification was carried out for 35 cycles, each comprising 45 sec at94° C., 45 sec at 52° C., and 1 min at 72° C. A PCR product ofapproximately 500 bp was excised from agarose gels, cloned and sequencedusing standard techniques. One plasmid clones were identified thatcontained a cloned PCR product of 517 bp. The nucleotide and deducedamino acid sequence of this PCR product are set forth as SEQ ID NO:177and SEQ ID NO:178. A BLASTP search of the annotated fungal proteindatabase at NCBI was conducted. Based on a CLUSTALW alignment, thededuced amino acid sequence of the PCR product shares 66.1% sequenceidentity to a hypothetical protein product from Gibberella zeae PH-1with GENBANK accession number XP 386864 which represents the proteinwith the closest similarity in this database. The protein has beenannotated as member of the MBOAT (membrane bound O-acyl transferase)family of membrane proteins that contains a variety of acyltransferaseenzymes.

Generation of a DGAT2 Specific Probe Using PCR

A DGAT2 gene fragment was generated by PCR as follows. The PCR reactioncontained 50 mM KCl, 10 mM Tris-HCl (pH 9), 0.1% Triton X-100, 2.5 mMMgCl₂, 0.2 mM each dNTP, 5 units of Taq polymerase (MBI Fermentas, USA),2 μl, of Lipomyces starkeyi cDNA and PCR primers P7 (SEQ ID NO:) and P8(SEQ ID NO:) at a final concentration of 1 μM. Amplification was carriedout for 35 cycles, each comprising 45 sec at 94° C., 45 sec at 52° C.,and 1 min at 72° C. A PCR product of approximately 270 bp was excisedfrom agarose gels, cloned and sequenced using standard techniques. Oneplasmid clones were identified that contained a cloned PCR product of263 bp. The nucleotide and deduced amino acid sequence of this PCRproduct are set forth as SEQ ID NO:179 and SEQ ID NO:180. A BLASTPsearch of the annotated fungal protein database at NCBI was conducted.Based on a CLUSTALW alignment, the deduced amino acid sequence of thePCR product shares 66.1% sequence identity to a hypothetical proteinproduct from Yarrowia lipolytica CLIB122 with GENBANK accession numberXP 504700 which represents the protein with the closest similarity inthis database. The amino acid sequence of this protein identical to thatof YL_DGAT 2 described in PCT Publication No. WO 2005/003322 and differsin one amino acid residue from that of YL-DGAT2 set forth as SEQ IDNO:10 for reasons that are outlined in Example 1.

Genomic DNA Isolation

A liquid culture (50 mL, YPD medium) of Lipomyces starkeyi was grown at28° C. for 72 h, at 250 rpm. Yeast cells were collected bycentrifugation and washed in deionized water. Yeast cells wereresuspended in 2 mL of STE buffer (0.1 M sorbitol, 10 mM Tris/HCl, pH7.5, 1 mM EDTA) containing 10 mg mL⁻¹ of Zymolase (Zymo ResearchCorporation, California, USA) and incubated for 2 hours at 37° C. Totalgenomic DNA was isolated from Lipomyces spheroplasts as follows. Theyeast suspension was lysed in 4 mL of Urea Extraction buffer (0.3125 MNaCl, 50 mM Tris HCl, pH 8.0, 20 mM EDTA, 1% sarkosine) and equal volumeof phenol/chloroform was added followed by thorough mixing. Aftercentrifugation the aqueous phase was re-extracted with phenolchloroform. Nucleic acids were precipitated by addition of ⅛ volume of4.4 M sodium acetate and 1 volume of isopropanol followed bycentrifugation. Nucleic acid were dried and resuspended in 500 microL TE(10 mM Tris, 1 mM EDTA). A small aliquot (1 microL) of the nucleic acidsolution was separated on 0.4% agarose gels next to molecular weightmarker to confirm that molecular weight was ≧50 kb.

Cosmid Library Construction

Genomic DNA of Lipomyces starkeyi was partially digested with MboI.Briefly, approximately 10 μg of genomic DNA were digested with 0.5 unitsof MboI (NEB, USA) in a final volume of 100 μL in the presence of 0.1 mgmL⁻¹ BSA, and 0.1 mg mL⁻¹ DNAse-free RNAse (Quiagen, USA) 100 mM NaCl,50 mM Tris-HCl, 10 mM MgCl₂, 1 mM DTT, pH 7.9. Aliquots of 25 μL wereremoved after 30, 60, 120 and 180 seconds respectively and combined in atube that contained 5 μL of 500 mM EDTA. The partially digested DNA waspurified and concentrated to a final volume of 10 μL using DNA Clean andConcentrator™ spin columns (Zymoresearch, USA) according to instructionsof the manufacturer. The DNA was ligated to 2 μg of linearized pLAFR3DNA (B. Staskawicz et al., Journal of bacteriology (1987), 169(12),5789-94) that had been completely digested with BamHI anddephosphorylated with Antarctic Phosphatase (NEB, USA). The ligationreaction was invitro-packaged and transfected into E. coli cells ofstrain NM554 (Stratagene, USA) using MAXPLAX packaging extracts(EPICENTRE Biotechnologies, USA) according to instructions of themanufacturer. Transfected cells were added to 15 mL of LB medium,incubated at 37° C. on Lab shaker set to 250 rpm. Glycerol was added toa final concentration of 15% (w/w) and the cell suspension was frozenusing a dry ice methanol mixture. The titer of the cosmid library wasdetermined by plating serial dilutions of the thawed cell suspension onsolidified LB medium containing 10 mg L⁻¹ of tetracycline.

Screening and Sequencing of Cosmids Comprising Lipomyces starkeyi DGAT1

Approximately 20,000 cosmid clones were plated on selective media,transferred to Biodyne B membranes (PALL Corporation, USA) and screenedwith a ³²P labeled probe corresponding to the cloned DGAT1-related PCRproduct of SEQ ID NO: derived from Lipomyces starkeyi according tostandard protocols. Cesium chloride density gradient centrifugation wasused as described in T. Maniatis et al. Molecular Cloning: A LaboratoryManual. (1982), 545 pp., was used to purify cosmid DNA from liquidcultures derived from colonies that hybridized to the probe understringent conditions. Two cosmid clones were sequenced using six primerswith SEQ ID NOs 181 through 188 (Table 44). Sequences were assembledusing the SEQMAN program of the LASERGENE™ 7.1.1 software package(DNASTAR, INC., USA). DNA sequencing of two independently isolatedcosmids with said primers produced DNA sequences that could be assembledinto a single sequence of 3343 bp referred to as LS_DGAT1 gene set forthas SEQ ID NO:189. A TBLASTX search of the annotated fungal proteindatabase at NCBI was conducted with the LS_DGAT 1 gene (SEQ ID NO:189)which showed similarities to hypothetical fungal proteins withsimilarities to acyltransferases. At also revealed that the LS_DGAT1gene is interrupted by introns. Therefore the PCR amplification andcloning of the LS DGAT1 cDNA was necessary. It is described in Example32.

TABLE 44 Primers used for sequencing of DGAT1 cosmid clonesderived from Lipomyces starkeyi SEQ ID Name Sequence NO: MWG713GTCAAGCGGATTCTGGAGATGGTGGGCC 181 MWG714 GGCCCACCATCTCCAGAATCCGCTTGAC 182MWG715 CCGCTTGTGCGCCGCGGGTGGAATTCTGCG 183 MWG716CGCAGAATTCCACCCGCGGCGCACAAGCGG 184 MWG717 CGCTGAGCGAAGCGACGTAG 185MWG718 CCCATTTTGGGGTTGGAGCG 186 MWG719 TTGATTTGCTTCTGGTCCCG 187 MWG720CGTGCCCATTGCATGGATTC 188Screening and Sequencing of Cosmids Comprising Lipomyces starkeyi DGAT2

Approximately 20,000 cosmid clones were plated on selective media,transferred to Biodyne B membranes (PALL Corporation, USA) and screenedwith a ³²P labeled probe corresponding to the cloned DGAT2-related PCRproduct of SEQ ID NO: derived from Lipomyces starkeyi according tostardard protocols. Cesium chloride density gradient centrifugation wasused as described in T. Maniatis et al. Molecular Cloning: A LaboratoryManual. (1982), 545 pp., was used to purify cosmid DNA from liquidcultures derived from colonies that hybridized to the probe understringent conditions. Four cosmid clones were sequenced using fourprimers with SEQ ID NOs 190 through 193 (Table 45). Sequences wereassembled using the SEQMAN program of the LASERGENE™ 7.1.1 softwarepackage (DNASTAR, INC., USA). DNA sequencing of four independentlyisolated cosmids with said primers produced DNA sequences that could beassembled into a single sequence of 2090 bp referred to as LS_DAGT2 geneset forth as SEQ ID NO:194. A TBLASTX search of the annotated fungalprotein database at NCBI was conducted with the LS_DGAT2 gene (SEQ IDNO:194) which showed similarities to hypothetical fungal proteins withsimilarities to acyltransferases. At also revealed that the LS_DGAT2gene is interrupted by introns. Therefore the PCR amplification andcloning of the LS DGAT2 cDNA was necessary. It is described in Example29.

TABLE 45 Primers used for sequencing of DGAT2 cosmid clonesderived from Lipomyces starkeyi SEQ ID Name Sequence NO: MWG667GGGTGCGTTTGGTGCTATTGGTACGGAAGG 190 MWG668 CCTTCCGTACCAATAGCACCAAACGCACCC191 MWG669 GGGATTGCTTCAGTATCTCGACGGTCTTG 192 MWG670CAAGACCGTCGAGATACTGAAGCAATCCC 193

Example 23 Cloning and Sequencing of DGAT2 from Rhodotorula glutinis

RNA Isolation and cDNA Synthesis

Rhodotorula glutinis (Catalog number 68-255T) was purchased from thePHAFF collection (UC Davis, Calif.). A liquid culture (50 mL, YPDmedium) of Rhodotorula glutinis was grown at 28° C. Cells were harvestedby centrifugation washed with deonized water and collected again bycentrifugation. Total RNA was isolated from the resulting cell culturepellet using the hot phenol method exactly as described in CurrentProtocols in Molecular Biology (John Wiley & Sons, Somerset, N.J.) butwhere all reagent volumes were increased by 10-fold (e.g. cell pelletwas re-suspended in 4 mL of TES solution instead of 400 μL). The finalRNA pellet was dissolved in 1 mL of water and the concentration wasdetermined to be 500 ng/uL.

cDNA was synthesized from 1 μg of total RNA (2 μl) using theSuperScript™ First-Strand Synthesis System for RT-PCR Kit (Invitrogen™Life Technologies, Carlsbad, Calif.) with the provided oligo(dT) primeraccording to the manufacturer's protocol. After RNase H treatment thecDNA was used as PCR template for generation of a DGAT2-specific genefragments as follows.

Generation of a DGAT2 Specific Probe Using PCR

A DGAT2 gene fragment was generated by PCR as follows. The PCR reactioncontained 50 mM KCl, 10 mM Tris-HCl (pH 9), 0.1% Triton X-100, 2.5 mMMgCl₂, 0.2 mM each dNTP, 5 units of Taq polymerase (MBI Fermentas, USA),2 μL of Rhodotorula glutinis cDNA and PCR primers BM DGAT2 FWD (SEQ IDNO:111) and BM DGAT2 REV (SEQ ID NO:112) at a final concentration of 1μM. Amplification was carried out for 35 cycles, each comprising 45 secat 94° C., 45 sec at 52° C., and 1 min at 72° C. A PCR product ofapproximately 480 bp was excised from agarose gels, cloned and sequencedusing standard techniques. Several plasmid clones were identified thatcontained a cloned PCR product of 474 bp. The nucleotide and deducedamino acid sequence of this PCR product are set forth as SEQ ID NO:195and SEQ ID NO:196. A BLASTP search of the annotated fungal proteindatabase at NCBI was conducted. Based on a CLUSTALW alignment, thededuced amino acid sequence of the PCR product shares 70.8% sequenceidentity to a hypothetical protein product from Ustilago maydis 521 withGENBANK accession number XP_(—)760084 which represents the protein withthe closest similarity in this database. This protein has been annotatedas having similarity to acyltransferases involved in phospholipidbiosynthesis and other proteins of unknown function.

Genomic DNA Isolation

A liquid culture (50 mL, YPD medium) of Rhodotorula glutinis was grownat 28° C. for 72 h, at 250 rpm. Yeast cells were collected bycentrifugation and washed in deionized water. Yeast cells wereresuspended in 2 mL of STE buffer (0.1 M sorbitol, 10 mM Tris/HCl, pH7.5, 1 mM EDTA) containing 10 mg mL⁻¹ of Zymolase (Zymo ResearchCorporation, California, USA) and incubated for 2 hours at 37° C. Totalgenomic DNA was isolated from Rhodotorula glutinis spheroplasts asfollows. The yeast suspension was lysed in 4 mL of Urea Extractionbuffer (0.3125 M NaCl, 50 mM Tris HCl, pH 8.0, 20 mM EDTA, 1% sarkosine)and equal volume of phenol/chloroform was added followed by thoroughmixing. After centrifugation the aqueous phase was re-extracted withphenol chloroform. Nucleic acids were precipitated by addition of ⅛volume of 4.4 M sodium acetate and 1 volume of isopropanol followed bycentrifugation. Nucleic acid were dried and resuspended in 500 microL TE(10 mM Tris, 1 mM EDTA). A small aliquot (1 microL) of the nucleic acidsolution was separated on 0.4% agarose gels next to molecular weightmarker to confirm that molecular weight was ≧50 kb.

Cosmid Library Construction

Genomic DNA of Rhodotorula glutinis was partially digested with MboI.Briefly, approximately 10 μg of genomic DNA were digested with 0.5 unitsof MboI (NEB, USA) in a final volume of 100 μL in the presence of 0.1 mgmL⁻¹ BSA, and 0.1 mg mL⁻¹ DNAse-free RNAse (Quiagen, USA) 100 mM NaCl,50 mM Tris-HCl, 10 mM MgCl₂, 1 mM DTT, pH 7.9. Aliquots of 25 μL wereremoved after 30, 60, 120 and 180 seconds respectively and combined in atube that contained 5 μL of 500 mM EDTA. The partially digested DNA waspurified and concentrated to a final volume of 10 μL using DNA Clean andConcentrator™ spin columns (Zymoresearch, USA) according to instructionsof the manufacturer. The DNA was ligated to 2 μg of linearized pLAFR3DNA (B. Staskawicz et al., Journal of bacteriology (1987), 169(12),5789-94) that had been completely digested with BamHI anddephosphorylated with Antarctic Phosphatase (NEB, USA). The ligationreaction was invitro-packaged and transfected into E. coli cells ofstrain NM554 (Stratagene, USA) using MAXPLAX packaging extracts(EPICENTRE Biotechnologies, USA) according to instructions of themanufacturer. Transfected cells were added to 15 mL of LB medium,incubated at 37° C. on Lab shaker set to 250 rpm. Glycerol was added toa final concentration of 15% (w/w) and the cell suspension was frozenusing a dry ice methanol mixture. The titer of the cosmid library wasdetermined by plating serial dilutions of the thawed cell suspension onsolidified LB medium containing 10 mg L⁻¹ of tetracycline.

Screening and Sequencing of Cosmids Comprising Rhodotorula glutinis DGAT2

Approximately 20,000 cosmid clones were plated on selective media,transferred to Biodyne B membranes (PALL Corporation, USA) and screenedwith a ³²P labeled probe corresponding to the cloned DGAT1-related PCRproduct of SEQ ID NO: derived from Rhodotorula glutinis according tostardard protocols. Cesium chloride density gradient centrifugation wasused as described in T. Maniatis et al. Molecular Cloning: A LaboratoryManual. (1982), 545 pp., was used to purify cosmid DNA from liquidcultures derived from colonies that hybridized to the probe understringent conditions. Seven cosmid clones were sequenced using sevenprimers with SEQ ID NOs 197 through 203 (Table 46). Sequences wereassembled using the SEQMAN program of the LASERGENE™ 7.1.1 softwarepackage (DNASTAR, INC., USA). DNA sequencing of seven independentlyisolated cosmids with said primers produced DNA sequences that could beassembled into a single sequence of 2944 bp referred to as RG_DGAT2 geneset forth as SEQ ID NO:204. A TBLASTX search of the annotated fungalprotein database at NCBI was conducted with the RG_DGAT2 gene (SEQ IDNO:204) which showed similarities to hypothetical fungal proteins withsimilarities to acyltransferases. At also revealed that the RG_DGAT2gene is interrupted by introns. Therefore the PCR amplification andcloning of the RG_DGAT2 cDNA was necessary. It is described in Example28.

TABLE 46 Primers used for sequencing of DGAT2 cosmid clonesderived from Rhodotorula glutinis SEQ ID Name Sequence NO: MWG649GCTTGATGAAGCCCTTGCGC 197 MWG650 GCGCAAGGGCTTCATCAAGC 198 MWG651GCCAAAGTTGGCGATGGCGC 199 MWG652 GCGCCATCGCCAACTTTGGC 200 MWG657GTGTGCGGCCTGAAACCGGG 201 MWG658 GCAGTGGGTGCATGGGTCAG 202 MWG659GAAGGCGACGATGCCGTGGC 203

Example 24 Cloning and Sequencing of DGAT2 from Phaffia rhodozyma

RNA Isolation and cDNA Synthesis

Phaffia rhodozyma (Catalog #67-210) was purchased from the PHAFFcollection at UC Davis, Calif., USA. A liquid culture (50 mL, YPDmedium) of Phaffia rhodozyma was grown at 18° C. Cells were harvested bycentrifugation washed with deonized water and collected again bycentrifugation. Total RNA was isolated from the resulting cell culturepellet using the hot phenol method exactly as described in CurrentProtocols in Molecular Biology (John Wiley & Sons, Somerset, N.J.) butwhere all reagent volumes were increased by 10-fold (e.g. cell pelletwas re-suspended in 4 mL of TES solution instead of 400 μL). The finalRNA pellet was dissolved in 1 mL of water and the concentration wasdetermined to be 500 ng/μL. cDNA was synthesized from 1 μg of total RNA(2 μl) using the SuperScript™ First-Strand Synthesis System for RT-PCRKit (Invitrogen™ Life Technologies, Carlsbad, Calif.) with the providedoligo(dT) primer according to the manufacturer's protocol. After RNase Htreatment the cDNA was used as PCR template for generation of aDGAT2-specific gene fragment as follows.

Generation of a DGAT2 Specific Probe Using PCR

A DGAT2 gene fragment was generated by PCR as follows. The PCR reactioncontained 50 mM KCl, 10 mM Tris-HCl (pH 9), 0.1% Triton X-100, 2.5 mMMgCl₂, 0.2 mM each dNTP, 5 units of Taq polymerase (MBI Fermentas, USA),2 μL of Phaffia rhodozyma cDNA and PCR primers BM DGAT2 FWD (SEQ IDNO:111) and BM DGAT2 REV (SEQ ID NO:112) at a final concentration of 1μM. Amplification was carried out for 35 cycles, each comprising 45 secat 94° C., 45 sec at 52° C., and 1 min at 72° C. A PCR product ofapproximately 400 bp was excised from agarose gels, cloned and sequencedusing standard techniques. One plasmid clones were identified thatcontained a cloned PCR product of 397 bp. The nucleotide and deducedamino acid sequence of this PCR product are set forth as SEQ ID NO:205and SEQ ID NO:206. A BLASTP search of the annotated fungal proteindatabase at NCBI was conducted. A CLUSTALW alignment revealed that thededuced amino acid sequence of the PCR product shares 77.1% sequenceidentity to a hypothetical protein product from Cryptococcus neoformansvar. neoformans B-3501A with GENBANK accession number XP_(—)774736 whichrepresents the protein with the closest similarity in this database.This protein has been annotated as having similarity to acyltransferasesinvolved in phospholipid biosynthesis and other proteins of unknownfunction.

TABLE 47 Primers used for 3′ and 5′ RACE amplification ofthe DGAT2 transcript derived from Phaffia rhodozyma SEQ ID Name SequenceNO: MWG709 GTCATCGGGATGGGAGCTTTCGCCAACTTTGC 207 MWG710GCAAAGTTGGCGAAAGCTCCCATCCCGATGAC 208 MWG711CCCGGGAACAGCCGACTTGACACTCAAGAG 209 MWG712 CTCTTGAGTGTCAAGTCGGCTGTTCCCGGG210 3′RACE GGCCACGCGTCGACTAGTACTTTTTTTTTTTTTT 211 3′RACEGGCCACGCGTCGACTAGTAC 212 ABR 5′RACE GGCCACGCGTCGACTAGTACGGGXXGGGXXGGXXG213 P Symbol (Meaning): R (G or A), Y (T or C), M (A or C), K (G or T),S (G or C), W (A or T), H (A or C or T), B (G or T or C), V (G or C orA), D (G or A or T), N (G or A or T or C), and X (Inosine)

3′ RACE

cDNA was synthesized from approximately 1 μg of total RNA of Phaffiarhodozyma (2 μl) using the SuperScript™ First-Strand Synthesis Systemfor RT-PCR Kit (Invitrogen™ Life Technologies, Carlsbad, Calif.) withthe 3′RACE primer (SEQ ID NO:) according to the manufacturer's protocol.After RNase H treatment the cDNA was used as PCR template for generationof a DGAT2-specific gene fragment as follows.

A DGAT2 gene fragment was generated by PCR as follows. The PCR reactioncontained 50 mM KCl, 10 mM Tris-HCl (pH 9), 0.1% Triton X-100, 2.5 mMMgCl₂, 0.2 mM each dNTP, 5 units of Taq polymerase (MBI Fermentas, USA),2 μl, of Phaffia rhodozyma cDNA generated with the 3′RACE primer (SEQ IDNO:211) and PCR primers MWG709 (SEQ ID NO:207) and 3′RACE ABR (SEQ IDNO:212) at a final concentration of 1 μM. Amplification was carried outfor 35 cycles, each comprising 45 sec at 94° C., 45 sec at 52° C., and 1min at 72° C. The resulting PCR reaction was diluted 1000 fold and 5 μLwere used in a PCR reaction using 3′RACE ABR primer (SEQ ID NO:212) andMWG 711 (SEQ ID NO:209). A PCR product of approximately 400 bp wasexcised from agarose gels and sequence directly using primer MWG 711(SEQ ID NO:209).

5′ RACE

cDNA was synthesized from 1 μg of total RNA of Phaffia rhodozyma (2 μL)using the SuperScript™ First-Strand Synthesis System for RT-PCR Kit(Invitrogen™ Life Technologies, Carlsbad, Calif.) with the MWG712 primer(SEQ ID NO:210) according to the manufacturer's protocol. After RNase Htreatment cytidyl residues were added to the 5′ end of the synthesizedcDNAs using recombinantly produced terminal transferase of calf thymus(New England Biolabs, Beverly, Mass.) according to manufacturer'sinstructions.

A DGAT2 gene fragment was generated by PCR as follows. The PCR reactioncontained 50 mM KCl, 10 mM Tris-HCl (pH 9), 0.1% Triton X-100, 2.5 mMMgCl₂, 0.2 mM each dNTP, 5 units of Taq polymerase (MBI Fermentas, USA),2 μl, of Phaffia rhodozyma C-tailed cDNA generated with the MWG712primer (SEQ ID NO:210) and PCR primers 5″RACE P (SEQ ID NO:213) andMWG710 (SEQ ID NO:208) at a final concentration of 1 μM. Amplificationwas carried out for 35 cycles, each comprising 45 sec at 94° C., 45 secat 52° C., and 1 min at 72° C. A PCR product of approximately 600 bp wasexcised from agarose gels and sequence directly using primer MWG710 (SEQID NO:208).

Assembly of a cDNA for PR DGAT2

Sequences derived from PCR products generated from total RNA using RTPCR and degenerate primers and 5′ and 3′ RACE were assembled using theSEQMAN program of the LASERGENE™ 7.1.1 software package (DNASTAR, INC.,USA). This produced a DNA sequence comprised of an ORF of 1218 bpencoding a protein of 405 amino acids. Nucleotide sequence and deducedamino acid sequence of this ORF which are referred to as PR_DGAT2 RACEand PR_DGAT2 PRO are set forth as SEQ ID NO:214 and SEQ ID:215. Based ona CLUSTALW alignment the deduced amino acid sequence of PR_DGAT2 RACEshares 60.2% sequence identity to a hypothetical protein product fromCryptococcus neoformans var. neoformans B-3501A with GENBANK accessionnumber XP_(—)774736 in a CLUSTALW alignment. This represents the proteinwith the closest similarity in the curated fungal protein database ofNCBI. It has been annotated as having similarity to acyltransferasesinvolved in phospholipid biosynthesis and other proteins of unknownfunction.

Example 25 Cloning and Sequencing of DGAT2 from Cryptococcus Curvatus

RNA Isolation and cDNA Synthesis Cryptococcus curvatus (ATCC 10567) waspurchased from the American Type Culture Collection (VA, USA). A liquidculture (50 mL, YPD medium) of Cryptococcus curvatus was grown at 28° C.Cells were harvested by centrifugation washed with deonized water andcollected again by centrifugation. Total RNA was isolated from theresulting cell culture pellet using the hot phenol method exactly asdescribed in Current Protocols in Molecular Biology (John Wiley & Sons,Somerset, N.J.) but where all reagent volumes were increased by 10-fold(e.g. cell pellet was re-suspended in 4 mL of TES solution instead of400 μL). The final RNA pellet was dissolved in 1 mL of water and theconcentration was determined to be 500 ng/μL. cDNA was synthesized from1 μg of total RNA (2 μl) using the SuperScript™ First-Strand SynthesisSystem for RT-PCR Kit (Invitrogen™ Life Technologies, Carlsbad, Calif.)with the provided oligo(dT) primer according to the manufacturer'sprotocol. After RNase H treatment the cDNA was used as PCR template forgeneration of a DGAT2-specific gene fragment as follows.

Generation of a DGAT2 Specific Probe Using PCR

A DGAT2 gene fragment was generated by PCR as follows. The PCR reactioncontained 50 mM KCl, 10 mM Tris-HCl (pH 9), 0.1% Triton X-100, 2.5 mMMgCl₂, 0.2 mM each dNTP, 5 units of Taq polymerase (MBI Fermentas, USA),2 μL of Cryptococcus curvatus cDNA and PCR primers P7 (SEQ ID NO:107)and P8 (SEQ ID NO:108) at a final concentration of 1 μM. Amplificationwas carried out for 35 cycles, each comprising 45 sec at 94° C., 45 secat 52° C., and 1 min at 72° C. A PCR product of approximately 270 bp wasexcised from agarose gels, cloned and sequenced using standardtechniques. One plasmid clones were identified that contained a clonedPCR product of 269 bp. The nucleotide and deduced amino acid sequence ofthis PCR product are set forth as SEQ ID NO:216 and SEQ ID NO:217. ABLASTP search of the annotated fungal protein database at NCBI wasconducted. Based on a CLUSTALW alignment, the deduced amino acidsequence of the PCR product shares 73.0% sequence identity to ahypothetical protein product from Cryptococcus neoformans var.neoformans B-3501A with GENBANK accession number XP_(—)774736 whichrepresents the protein with the closest similarity in this database.This protein has been annotated as having similarity to acyltransferasesinvolved in phospholipid biosynthesis and other proteins of unknownfunction.

Genomic DNA Isolation

A liquid culture (50 mL, YPD medium) of Cryptococcus curvatus was grownat 28° C. for 72 h, at 250 rpm. Yeast cells were collected bycentrifugation and washed in deionized water. Yeast cells wereresuspended in 2 mL of STE buffer (0.1 M sorbitol, 10 mM Tris/HCl, pH7.5, 1 mM EDTA) containing 10 mg mL⁻¹ of Zymolase (Zymo ResearchCorporation, California, USA) and incubated for 2 hours at 37° C. Totalgenomic DNA was isolated from Cryptococcus curvatus spheroplasts asfollows. The yeast suspension was lysed in 4 mL of Urea Extractionbuffer (0.3125 M NaCl, 50 mM Tris HCl, pH 8.0, 20 mM EDTA, 1% sarkosine)and equal volume of phenol/chloroform was added followed by thoroughmixing. After centrifugation the aqueous phase was re-extracted withphenol chloroform. Nucleic acids were precipitated by addition of ⅛volume of 4.4 M sodium acetate and 1 volume of isopropanol followed bycentrifugation. Nucleic acid were dried and resuspended in 500 microL TE(10 mM Tris, 1 mM EDTA). A small aliquot (1 microL) of the nucleic acidsolution was separated on 0.4% agarose gels next to molecular weightmarker to confirm that molecular weight was ≧50 kb.

Cosmid Library Construction

Genomic DNA of Cryptococcus curvatus was partially digested with MboI.Briefly, approximately 10 μg of genomic DNA were digested with 0.5 unitsof MboI (NEB, USA) in a final volume of 100 μL in the presence of 0.1 mgmL⁻¹ BSA, and 0.1 mg mL^(−1 DNAse-free RNAse (Quiagen, USA)) 100 mMNaCl, 50 mM Tris-HCl, 10 mM MgCl₂, 1 mM DTT, pH 7.9. Aliquots of 25 μLwere removed after 30, 60, 120 and 180 seconds respectively and combinedin a tube that contained 5 μL of 500 mM EDTA. The partially digested DNAwas purified and concentrated to a final volume of 10 μL it using DNAClean and Concentrator™ spin columns (Zymoresearch, USA) according toinstructions of the manufacturer. The DNA was ligated to 2 μg oflinearized pLAFR3DNA (B. Staskawicz et al., Journal of bacteriology(1987), 169(12), 5789-94) that had been completely digested with BamHIand dephosphorylated with Antarctic Phosphatase (NEB, USA). The ligationreaction was invitro-packaged and transfected into E. coli cells ofstrain NM554 (Stratagene, USA) using MAXPLAX packaging extracts(EPICENTRE Biotechnologies, USA) according to instructions of themanufacturer. Transfected cells were added to 15 mL of LB medium,incubated at 37° C. on Lab shaker set to 250 rpm. Glycerol was added toa final concentration of 15% (w/w) and the cell suspension was frozenusing a dry ice methanol mixture. The titer of the cosmid library wasdetermined by plating serial dilutions of the thawed cell suspension onsolidified LB medium containing 10 mg L⁻¹ of tetracycline.

Screening and sequencing of cosmids comprising Cryptococcus curvatusDgat2

Approximately 20,000 cosmid clones were plated on selective media,transferred to Biodyne B membranes (PALL Corporation, USA) and screenedwith a ³²P labeled probe corresponding to the cloned DGAT2-related PCRproduct of SEQ ID NO:216 derived from Cryptococcus curvatus according tostandard protocols. Cesium chloride density gradient centrifugation wasused as described in T. Maniatis et al. Molecular Cloning: A LaboratoryManual. (1982), 545 pp., was used to purify cosmid DNA from liquidcultures derived from colonies that hybridized to the probe understringent conditions. Four cosmid clones were sequenced using eightprimers with SEQ ID NOs 218 through 225 (Table 48). Sequences wereassembled using the SEQMAN program of the LASERGENE™ 7.1.1 softwarepackage (DNASTAR, INC., USA). DNA sequencing of seven independentlyisolated cosmids with said primers produced DNA sequences that could beassembled into a single sequence of 2816 bp referred to as CC_DGAT2 geneset forth as SEQ ID NO:226. A TBLASTX search of the annotated fungalprotein database at NCBI was conducted with the CC_DGAT2 gene (SEQ IDNO:226) which showed similarities to hypothetical fungal proteins withsimilarities to acyltransferases. At also revealed that the CC_DGAT2gene is interrupted by several introns.

TABLE 48 Primers used for sequencing of DGAT2 cosmid clonesderived from Cryptococcus curvatus SEQ ID Name Sequence NO: MWG732GGCATGGGAGCTGTCGCGGCCTTTGCGTCCGAGGC 218 MWG733GCCTCGGACGCAAAGGCCGCGACAGCTCCCATGCC 219 MWG734CCTGGCCAAGGGTCCGGGCTATGCCATCACC 220 MWG735GGTGATGGCATAGCCCGGACCCTTGGCCAGG 221 MWG743GCGGCGTGCGGTTGTGGTCGTACCCGGCCC 222 MWG744 CCCAGATCATGTACGGGATGAGCACTGGC223 MWG745 CCTTCCGCCACCCCATCGTCACTGTCG 224 MWG746CGCTCTTCCACGGGCGCGGGCTCTTCAAC 225

Example 26 Cloning and Sequencing of DGAT2 from Mucor circinelloides

RNA Isolation and cDNA Synthesis

Mucor circinelloides (ATCC 1216b) was purchased from the American TypeCulture Collection (VA, USA). A liquid culture (50 mL of potato dextrosebroth) of Mucor circinelloides was grown at 24° C. Potato dextrose brothwas purchased from Becton, Dickinson and Company (NJ, USA). The myceliumwas harvested by centrifugation washed with deionized water andcollected again by centrifugation. Total RNA was isolated from theresulting mycelium using the hot phenol method exactly as described inCurrent Protocols in Molecular Biology (John Wiley & Sons, Somerset,N.J.) but where all reagent volumes were increased by 10-fold (e.g. cellpellet was re-suspended in 4 mL of TES solution instead of 400 mL). Thefinal RNA pellet was dissolved in 1 mL of water and the concentrationwas determined to be 500 ng/uL. cDNA was synthesized from 1 ng of totalRNA (2 μl) using the SuperScript™ First-Strand Synthesis System forRT-PCR Kit (Invitrogen™ Life Technologies, Carlsbad, Calif.) with theprovided oligo(dT) primer according to the manufacturer's protocol.After RNase H treatment the cDNA was used as PCR template for generationof a DGAT2-specific gene fragment as follows.

Generation of a DGAT2 Specific Probe Using PCR

A DGAT2 gene fragment was generated by PCR as follows. The PCR reactioncontained 50 mM KCl, 10 mM Tris-HCl (pH 9), 0.1% Triton X-100, 2.5 mMMgCl₂, 0.2 mM each dNTP, 5 units of Taq polymerase (MBI Fermentas, USA),2 μL of Mucor circinelloides cDNA and PCR primers BM DGAT2 FWD (SEQ IDNO:) and BM DGAT2 REV (SEQ ID NO:) at a final concentration of 1 μM.Amplification was carried out for 35 cycles, each comprising 45 sec at94° C., 45 sec at 52° C., and 1 min at 72° C. A PCR product ofapproximately 400 bp was excised from agarose gels, cloned and sequencedusing standard techniques. One plasmid clones were identified thatcontained a cloned PCR product of 394 bp. The nucleotide and deducedamino acid sequence of this PCR product are set forth as SEQ ID NO:227and SEQ ID NO:228. A BLASTP search of the annotated fungal proteindatabase at NCBI was conducted. The deduced amino acid sequence of thePCR product shares 67.2% sequence identity in a CLUSTALW alignment to ahypothetical protein product from Cryptococcus neoformans var.neoformans B-3501A with GENBANK accession number XP 774736 whichrepresents the protein with the closest similarity in the curated fungalprotein database at NCBI. This protein has been annotated as havingsimilarity to acyltransferases involved in phospholipid biosynthesis andother proteins of unknown function.

TABLE 49 Primers used for 3′ and 5′ RACE amplification ofthe DGAT2 transcript derived from Mucor circinelloides SEQ ID NameSequence NO: MWG736 GCAGTTTTGCTAGTTTTGCTACGGAAGC 229 MWG737GCTTCCGTAGCAAAACTAGCAAAACTGC 230 MWG738 GAGATTAGGTTTCATCCGAATCGCGATTCGTC231 MWG739 GACGAATCGCGATTCGGATGAAACCTAATCTC 232 3′RACEGGCCACGCGTCGACTAGTACTTTTTTTTTTTTTT 211 3′RACE GGCCACGCGTCGACTAGTAC 212ABR 5′RACE GGCCACGCGTCGACTAGTACGGGXXGGGXXGGGXXG 213 P Symbol (Meaning):R (G or A), Y (T or C), M (A or C), K (G or T), S (G or C), W (A or T),H (A or C or T), B (G or T or C), V (G or C or A), D (G or A or T), N (Gor A or T or C), and X (Inosine)

3′ RACE

cDNA was synthesized from 1 μg of total RNA of Mucor circinelloides (2μL) using the SuperScript™ First-Strand Synthesis System for RT-PCR Kit(Invitrogen™ Life Technologies, Carlsbad, Calif.) with the 3′RACE primer(SEQ ID NO:) according to the manufacturer's protocol. After RNase Htreatment the cDNA was used as PCR template for generation of aDGAT2-specific gene fragment as follows.

A DGAT2 gene fragment was generated by PCR as follows. The PCR reactioncontained 50 mM KCl, 10 mM Tris-HCl (pH 9), 0.1% Triton X-100, 2.5 mMMgCl₂, 0.2 mM each dNTP, 5 units of Taq polymerase (MBI Fermentas, USA),2 μL of Mucor circinelloides cDNA generated with the 3′RACE primer (SEQID NO:211) and PCR primers MWG736 (SEQ ID NO:229) and 3′RACE ABR primer(SEQ ID NO:212) at a final concentration of 1 μM. Amplification wascarried out for 35 cycles, each comprising 45 sec at 94° C., 45 sec at52° C., and 1 min at 72° C. A prominent PCR product of approximately 650bp was excised form agarose gels and directly sequenced with the MWG736(SEQ ID NO:229) and MWG738 (SEQ ID NO:231) primers.

5′ RACE

cDNA was synthesized from 1 μg of total RNA of Mucor circinelloides (2μl) using the SuperScript™ First-Strand Synthesis System for RT-PCR Kit(Invitrogen™ Life Technologies, Carlsbad, Calif.) with the MWG739 primer(SEQ ID NO:232) according to the manufacturer's protocol. After RNase Htreatment cytidyl residues were added to the 5′ end of the synthesizedcDNAs using recombinantly produced terminal transferase of calf thymus(New England Biolabs, Beverly, Mass.) according to manufacturer'sinstructions.

A DGAT2 gene fragment was generated by PCR as follows. The PCR reactioncontained 50 mM KCl, 10 mM Tris-HCl (pH 9), 0.1% Triton X-100, 2.5 mMMgCl₂, 0.2 mM each dNTP, 5 units of Taq polymerase (MBI Fermentas, USA),2 μL of Mucor circinelloides C-tailed cDNA generated with the MWG739primer (SEQ ID NO:232) and PCR primers 5′RACE P (SEQ ID NO:213) andMWG737 (SEQ ID NO:230) at a final concentration of 1 μM. Amplificationwas carried out for 35 cycles, each comprising 45 sec at 94° C., 45 secat 52° C., and 1 min at 72° C. A PCR product of approximately 600 bp wasexcised from agarose gels and cloned into a plasmid vector. Two plasmidclones with an insert of approximately 520 bp were sequenced completely.

Assembly of a cDNA for MC_DGAT2

DNA sequences derived from PCR products generated from total RNA usingRT PCR and degenerate primers and 5′ and 3′ RACE were assembled usingthe SEQMAN program of the LASERGENE™ 7.1.1 software package (DNASTAR,INC., USA). This produced a DNA sequence comprised of an ORF of 1110 bpencoding a protein of 369 amino acids. Nucleotide sequence and deducedamino acid sequence of this ORF referred to as MC_DGAT2 are set forth asSEQ ID NO:233 and SEQ ID:234. Based on a CLUSTALW alignment, the deducedamino acid sequence of MC_DGAT2 shares 49.7% sequence identity to ahypothetical protein product from Ustilago maydis 521 with GENBANKaccession number XP 760084 which represents the protein with the closestsimilarity in the curated fungal protein database at NCBI. This proteinhas been annotated as having similarity to acyltransferases involved inphospholipid biosynthesis and other proteins of unknown function. Theprotein also shares 54.4 and 59.7% sequence identity to the DGAT2a andDAGT2b proteins of Mortierella ramanniana described in Lardizabal et al(The Journal of biological chemistry (2001), 276(42), 38862-9.)

Example 27 Expression of DGAT2s from Yarrowia lipolyitica, Torulosporadelbrueckii and Debaryomyces hansenii in Soybean Somatic Embryos

The present example describes construction of pKR1324, comprisingTorulospora diacylglyceride acyltransferase 2a (TD_DGAT2a); pKR1325,comprising Torulospora diacylglyceride acyltransferase 2b (TD_DGAT2b);and pKR1328, comprising Debaryomyces diacylglyceride acyltransferase 2(DH_DGAT2) and expression in somatic embryos. Control vector pKR1256(SEQ ID NO:98; Example 16), comprising Yarrowia DGAT2, was alsoexpressed for comparison.

Construction of pKR1324, comprising TD DGAT2a

TD DGAT2a (SEQ ID NO:121) was amplified from a Torulospora cosmid clonedescribed in Example 18 with oligonucleotide primers ocgDG2a-1 (SEQ IDNO:235) and ocgDG2a-2 (SEQ ID NO:236), using the Phusion™ High-FidelityDNA Polymerase (Cat. No. F553S, Finnzymes Oy, Finland) following themanufacturer's protocol. The resulting DNA fragment was cloned into thepCR-Blunt® cloning vector using the Zero Blunt® PCR Cloning Kit(Invitrogen Corporation), following the manufacturer's protocol, toproduce pKR1295 (SEQ ID NO:237). The nucleotide and corresponding aminoacid sequence of TD DGAT2a from pKR1295 is set forth in SEQ ID NO:133and SEQ ID NO:135.

The NotI fragment of pKR1295 (SEQ ID NO:237), containing TD DGAT2a, wascloned into the NotI site of pKR72 (SEQ ID NO:26; Example 4) to producepKR1324 (SEQ ID NO:238).

Construction of pKR1325, comprising TD DGAT2b

TD DGAT2b (SEQ ID NO:134) was amplified from Torulospora cosmid DNAdescribed in Example 18 with oligonucleotide primers ocgDG2b-1 (SEQ IDNO:239) and ocgDG2b-2 (SEQ ID NO:240), using the Phusion™ High-FidelityDNA Polymerase (Cat. No. F553S, Finnzymes Oy, Finland) following themanufacturer's protocol. The resulting DNA fragment was cloned into thepCR-Blunt® cloning vector using the Zero Blunt® PCR Cloning Kit(Invitrogen Corporation), following the manufacturer's protocol, toproduce pKR1296 (SEQ ID NO:241). The nucleotide and corresponding aminoacid sequence of TD DGAT2b from pKR1296 is set forth in SEQ ID NO:134and SEQ ID NO:136.

The NotI fragment of pKR1296 (SEQ ID NO:241), containing TD DGAT2b, wascloned into the NotI site of pKR72 (SEQ ID NO:26; Example 4) to producepKR1325 (SEQ ID NO:242).

Construction of pKR1328, comprising DH DGAT2

DH DGAT2 (SEQ ID NO:161) was amplified from a Debaryomyces cosmid DNAclone described in Example 20 with oligonucleotide primers odhDG2-1 (SEQID NO:243) and odhDG2-2 (SEQ ID NO:244), using the Phusion™High-Fidelity DNA Polymerase (Cat. No. F553S, Finnzymes Oy, Finland)following the manufacturer's protocol. The resulting DNA fragment wascloned into the pCR-Blunt® cloning vector using the Zero Blunt® PCRCloning Kit (Invitrogen Corporation), following the manufacturer'sprotocol, to produce pKR1297 (SEQ ID NO:245). The nucleotide andcorresponding amino acid sequence of DH DGAT2 from pKR1297 is set forthin SEQ ID NO:161 and SEQ ID NO:162.

The NotI fragment of pKR1297 (SEQ ID NO:245), containing DH DGAT2, wascloned into the NotI site of pKR179 (SEQ ID NO:246), which is describedin BB1574 US 20080095915 (the contents of which are incorporated byreference) to produce pKR1327 (SEQ ID NO:247).

The PstI fragment of pKR1327 (SEQ ID NO:247), containing DH DGAT2, wascloned into the SbfI site of pKR325 (SEQ ID NO:248), previouslydescribed in PCT Publication No. WO 2006/012325 (the contents of whichare hereby incorporated by reference) to produce pKR1328 (SEQ IDNO:249).

Expression of TD DGAT2a, TD DGAT2b and DH DGAT2 in Soy Somatic Embryos

Soybean embryogenic suspension culture (cv. Jack) was transformed withpKR1324

(SEQ ID NO:238), having experiment number MSE2268; with pKR1325 (SEQ IDNO:242), having experiment number MSE2269 and with pKR1328 (SEQ IDNO:249), having experiment number MSE2270. Control vector pKR1256 (SEQID NO:98; Example 16), comprising Yarrowia DGAT2, was also transformedalone in a similar way for a control and has experiment number MSE2267.Events were selected and somatic embryos matured in SHaM as described inExample 5. Oil concentrations and fatty acid profiles were determined asdescribed in Example 5 for MSE2267, MSE2268, MSE 2269 and MSE2270 andresults for each experiment are shown in Table 50, Table 51, Table 52and Table 53, respectively.

TABLE 50 Oil concentrations and fatty acid profiles for events fromMSE2267 MSE2267 (YL DGAT2) Event 16:0 18:0 18:1 18:2 18:3 % Oil 2267-1314.4 7.2 33.1 36.8 8.6 10.6 2267-16 12.9 7.5 34.9 35.9 8.9 10.2 2267-2113.9 8.2 33.5 35.3 9.1 9.6 2267-26 12.3 8.9 33.2 36.8 8.8 9.6 2267-815.9 8.3 25.6 37.4 12.7 9.0 2267-18 14.5 7.7 31.1 36.1 10.6 8.4 2267-1915.4 7.5 24.4 40.5 12.2 8.2 2267-5 15.5 7.4 24.5 40.8 11.7 8.1 2267-3014.3 6.4 27.3 39.2 12.8 8.0 2267-4 15.4 6.4 22.0 43.4 12.8 7.8 2267-916.7 8.0 25.8 36.2 13.3 7.7 2267-31 15.8 8.6 27.6 36.3 11.7 7.7 2267-2015.2 7.2 27.1 38.1 12.4 7.6 2267-24 16.7 7.8 23.9 40.7 10.9 7.5 2267-1516.6 7.6 22.2 41.8 11.8 7.4 2267-3 15.8 8.1 29.3 36.2 10.6 7.4 2267-2914.8 9.0 28.8 35.5 11.9 7.3 2267-25- 16.3 8.9 26.1 36.2 12.5 7.1 2267-1015.8 8.3 25.8 36.9 13.2 7.0 2267-14 17.2 6.6 17.9 44.2 14.1 6.9 2267-1716.9 7.1 22.1 41.3 12.6 6.9 2267-23 17.3 5.8 18.4 41.7 16.7 6.6 2267-2216.8 7.9 24.2 38.3 12.8 6.5 2267-11 15.3 5.0 20.6 47.8 11.3 6.5 2267-2817.2 7.2 21.6 39.6 14.4 6.3 2267-7 15.0 7.9 27.2 37.5 12.4 5.8 2267-1218.1 5.9 19.0 42.1 15.0 5.7 2267-6 16.5 5.9 19.7 40.2 17.7 5.4 2267-2716.0 6.7 18.8 40.8 17.6 5.0 2267-2 18.3 7.6 23.3 35.6 15.3 4.6 2267-118.6 5.7 17.7 41.4 16.6 4.6 Avg. 15.9 7.4 25.1 39.1 12.7 7.3

TABLE 51 Oil concentrations and fatty acid profiles for events fromMSE2268 MSE2268 (TD DGAT2a) Event 16:0 18:0 18:1 18:2 18:3 % Oil 2268-115.3 6.0 23.2 44.2 11.3 9.6 2268-30 16.7 5.2 19.5 45.5 13.0 9.3 2268-2315.2 7.4 23.9 42.3 11.3 9.0 2268-4 15.3 5.4 22.8 43.6 12.8 8.8 2268-1315.2 6.8 25.1 41.4 11.5 8.8 2268-21 16.0 7.0 22.6 41.5 12.9 8.3 2268-2615.8 7.2 22.4 41.6 13.1 7.7 2268-27 16.5 5.0 18.4 45.9 14.3 7.4 2268-615.7 7.5 24.0 40.4 12.4 7.4 2268-16 16.6 6.5 22.6 40.3 14.0 7.4 2268-816.4 6.6 22.2 42.5 12.2 7.0 2268-2 16.6 5.0 16.8 45.3 16.2 6.6 2268-1816.0 5.4 20.0 43.7 14.8 6.5 2268-14 16.5 5.8 19.5 42.4 15.8 6.5 2268-1216.6 6.5 21.4 41.1 14.5 6.4 2268-10 16.5 6.8 22.6 40.2 13.9 6.3 2268-1917.3 6.7 22.7 40.3 13.0 6.2 2268-20 16.8 7.5 20.8 41.9 13.0 6.2 2268-2816.5 6.5 18.6 44.7 13.6 6.1 2268-11 15.7 5.8 21.0 41.2 16.3 5.6 2268-716.2 6.7 22.5 39.9 14.7 5.6 2268-22 15.8 7.1 25.0 37.4 14.6 5.5 2268-317.2 6.8 20.4 41.2 14.3 5.5 2268-29 15.8 7.0 24.2 38.6 14.4 5.4 2268-1516.6 6.6 23.1 39.5 14.2 5.3 2268-5 17.1 6.6 20.1 41.8 14.4 5.2 2268-1716.6 5.0 18.2 42.6 17.6 5.0 2268-25 17.7 5.7 17.5 41.1 18.0 4.6 2268-2418.6 5.4 17.7 41.1 17.3 4.4 2268-31 18.0 5.9 21.0 37.6 17.6 3.6 2268-918.7 5.6 17.2 40.2 18.3 3.5 Avg. 16.5 6.3 21.2 41.7 14.4 6.5

TABLE 52 Oil concentrations and fatty acid profiles for events fromMSE2269 MSE2269 (TD DGAT2b) Event 16:0 18:0 18:1 18:2 18:3 % Oil 2269-2015.2 5.2 20.0 48.5 11.1 10.8 2269-15 15.0 6.0 21.1 45.1 12.7 9.6 2269-414.4 5.4 21.9 46.5 11.8 9.6 2269-17 15.8 5.3 21.9 45.7 11.3 9.5 2269-1416.2 5.8 20.1 44.7 13.3 8.5 2269-27 16.3 5.6 20.1 45.6 12.4 8.3 2269-616.4 4.7 18.0 46.4 14.5 8.1 2269-24 16.2 5.9 21.8 43.0 13.1 8.0 2269-1616.6 6.5 18.9 43.9 14.2 7.8 2269-11 15.7 7.7 25.0 39.5 12.1 7.8 2269-1315.9 6.1 23.3 41.6 13.1 7.6 2269-19 16.2 5.9 21.4 43.0 13.6 7.4 2269-716.1 5.3 20.2 43.6 14.8 7.3 2269-1 16.4 7.1 24.7 39.1 12.8 7.2 2269-2616.7 5.0 19.6 43.5 15.1 7.0 2269-10 17.0 5.3 17.7 43.9 16.1 6.6 2269-2116.3 7.0 23.6 38.3 14.8 6.6 2269-18 16.7 5.7 19.7 42.8 15.1 6.5 2269-317.0 5.4 19.6 41.4 16.7 6.1 2269-5 17.6 5.0 17.9 43.4 16.1 6.0 2269-2817.3 6.6 17.6 43.2 15.3 5.6 2269-8 16.7 4.8 18.3 43.5 16.7 5.4 2269-2516.4 4.9 16.9 45.1 16.6 5.1 2269-23 17.9 5.6 18.1 41.5 17.0 5.1 2269-916.6 5.4 17.9 42.4 17.6 4.9 2269-31 17.0 5.6 16.4 43.5 17.6 4.8 2269-218.0 4.3 14.6 42.3 20.8 3.5 2269-22 18.8 5.4 15.6 40.9 19.3 3.5 2269-1218.1 5.4 15.4 42.3 18.8 3.3 2269-30 19.1 4.8 15.0 41.1 20.0 3.1 2269-2919.2 4.9 14.3 42.9 18.7 2.9 Avg. 16.7 5.6 19.2 43.2 15.3 6.6

TABLE 53 Oil concentrations and fatty acid profiles for events fromMSE2270 MSE2270 (DH DGAT2) Event 16:0 18:0 18:1 18:2 18:3 % Oil 2270-2215.7 4.8 20.7 47.1 11.7 10.6 2270-20 15.5 4.9 20.4 46.3 12.9 9.5 2270-816.7 5.2 20.5 44.6 13.0 8.9 2270-29 17.1 5.6 19.6 41.8 15.9 8.1 2270-3015.9 6.2 23.5 41.8 12.6 7.9 2270-21 16.9 5.5 19.8 41.8 16.0 7.2 2270-217.0 5.6 20.6 42.2 14.6 7.1 2270-20 16.7 5.7 21.4 42.1 14.1 7.1 2270-216.9 5.9 19.2 42.9 15.1 6.7 2270-4 16.8 6.9 20.5 40.6 15.2 6.6 2270-2716.0 6.2 21.5 42.1 14.2 6.3 2270-26 16.4 5.6 20.4 42.0 15.7 6.2 2270-916.3 4.7 17.7 44.7 16.5 6.2 2270-28 17.1 4.8 17.7 44.2 16.2 6.0 2270-2118.6 5.8 16.9 41.3 17.4 5.6 2270-26 18.7 5.3 18.2 39.5 18.2 5.3 2270-2217.4 6.2 18.6 39.2 18.5 5.1 2270-23 19.5 4.7 17.0 42.3 16.5 4.9 2270-2419.4 5.0 15.2 42.1 18.3 4.8 2270-28 16.2 5.2 20.1 45.1 13.4 4.6 2270-2516.0 8.0 20.6 39.1 16.1 4.6 2270-31 18.0 5.0 16.7 41.5 18.8 4.5 2270-2517.1 5.4 18.7 40.9 17.9 4.2 2270-24 13.9 5.2 16.3 45.7 18.9 4.1 2270-518.0 6.2 20.6 38.2 16.9 4.0 2270-23 19.6 4.5 15.0 40.6 20.3 4.0 2270-2717.8 5.1 16.7 41.9 18.4 3.8 2270-6 17.1 5.7 16.8 41.5 18.9 3.5 2270-318.5 5.3 17.0 40.7 18.6 3.5 2270-7 17.3 4.3 14.4 40.9 23.2 3.2 2270-2918.7 4.1 12.4 40.4 24.3 2.5 Avg. 17.2 5.4 18.5 42.1 16.7 5.7

TD_DGAT2a, TD_DGAT2b and DH_DGAT2 increase oil concentrations in somaticembryos to amounts similar to YL_DGAT2. But, in contrast to what is seenfor YL_DGAT2 (i.e. increasing oleic acid concentrations, decreasingpalmitic acid and alpha-linolenic acid concentrations) effects on fattyacid profiles are not as strong for TD_DGAT2a, TD_DGAT2b and DH_DGAT2.

Example 28 Expression of DGAT2s from Yarrowia lipolyitica, Mortierellaalpina, Pichia anomala and Rhodotorula glutinis in Soybean SomaticEmbryos

The present example describes construction of pKR1335, comprisingMortierella alpina diacylglyceride acyltransferase 2 (Ma_DGAT2);pKR1332, comprising Pichia anomala diacylglyceride acyltransferase 2(PA_DGAT2); and pKR1333, comprising Rhodorurola glutinis diacylglycerideacyltransferase 2 (RG_DGAT2) and expression in somatic embryos. Controlvector pKR1256 (SEQ ID NO:98; Example 16), comprising Yarrowia DGAT2,was also expressed for comparison.

Construction of pKR1335, comprising MA DGAT2

MA_DGAT2 was amplified from pMDGAT2-17 (SEQ ID NO:250), which isdescribed in issued U.S. Pat. No. 7,198,937-CRD MalpinaDGAT2 patent (thecontents of which is incorporated by reference) with oligonucleotideprimers oMaDG2-3 (SEQ ID NO:251) and oMaDG2-4 (SEQ ID NO:252), using thePhusion™ High-Fidelity DNA Polymerase (Cat. No. F553S, Finnzymes Oy,Finland) following the manufacturer's protocol. The resulting DNAfragment was cloned into the pCR-Blunt® cloning vector using the ZeroBlunt® PCR Cloning Kit (Invitrogen Corporation), following themanufacturer's protocol, to produce pKR1330 (SEQ ID NO:253). Thenucleotide and corresponding amino acid sequence of MA DGAT2 frompKR1330 is set forth in SEQ ID NO:254 and SEQ ID NO:255.

The NotI fragment of pKR1330 (SEQ ID NO:253), containing MA DGAT2, wascloned into the NotI site of pKR72 (SEQ ID NO:26; Example 4) to producepKR1335 (SEQ ID NO:256).

Construction of pKR1332, comprising PA DGAT2

PA DGAT2 (SEQ ID NO:146) was amplified from a Pichia anomala cosmid DNAclone described in Example 19 with oligonucleotide primers oTdDG2-1 (PA)(SEQ ID NO:257) and oTdDG2-2(PA) (SEQ ID NO:258), using the Phusion™High-Fidelity DNA Polymerase (Cat. No. F553S, Finnzymes Oy, Finland)following the manufacturer's protocol. The resulting DNA fragment wascloned into the pCR-Blunt® cloning vector using the Zero Blunt® PCRCloning Kit (Invitrogen Corporation), following the manufacturer'sprotocol, to produce pKR1319 (SEQ ID NO:259). The nucleotide andcorresponding amino acid sequence of PA_DGAT2 from pKR1319 is set forthin SEQ ID NO:146 and SEQ ID NO:147

The NotI fragment of pKR1319 (SEQ ID NO:259), containing PA DGAT2, wascloned into the NotI site of pKR72 (SEQ ID NO:26; Example 4) to producepKR1332 (SEQ ID NO:260).

Construction of pKR1333, comprising RG DGAT2

Because the RG DGAT2 genomic sequence (SEQ ID NO:204; Example 23)appeared to contain numerous introns when compared to other DGATs byBlastX analysis, the RG DGAT2 coding sequence was cloned by PCR fromcDNA and RNA isolation and cDNA synthesis is described below.

Oligonucleotide primers RgCDNA-5 (SEQ ID NO:261) and RgCDNA-3 (SEQ IDNO:262), which were designed based on the genomic DNA sequence of theRG_DGAT2 gene (SEQ ID NO:204; Example 23). Using the Phusion™High-Fidelity DNA Polymerase (Cat. No. F553S, Finnzymes Oy, Finland)following the manufacturer's protocol the RG_DGAT2 transcript wasamplified from cDNA of Rhodotorula glutinis that was generated asdescribed in Example 23. The resulting DNA fragment was cloned into thepCR-Blunt® cloning vector using the Zero Blunt® PCR Cloning Kit(Invitrogen Corporation), following the manufacturer's protocol, toproduce pHD28 (SEQ ID NO:263). The RG DGAT2 cDNA nucleotide andcorresponding amino acid sequences are set forth in SEQ ID NO:264 andSEQ ID NO:265, respectively. A BLASTP search of the annotated fungalprotein database at NCBI was conducted. Based on a CLUSTALW aligment,the deduced amino acid sequence of the RG_DGAT2 transcript shares 62.4%sequence identity to a hypothetical protein product from Ustilago maydis521 with GENBANK accession number XP_(—)760084 which represents theprotein with the closest similarity in this database. This protein hasbeen annotated as having similarity to acyltransferases involved inphospholipid biosynthesis and other proteins of unknown function.

The NotI fragment of pHD28 (SEQ ID NO:263), containing RG_DGAT2, wascloned into the NotI site of pKR72 (SEQ ID NO:26; Example 4) to producepKR1333 (SEQ ID NO:266).

Expression of Ma_DGAT2, PA DGAT2 and RG DGAT2 in Soy Somatic Embryos

Soybean embryogenic suspension culture (cv. Jack) was transformed withpKR1335 (SEQ ID NO:256), having experiment number MSE2296; with pKR1332(SEQ ID NO:260), having experiment number MSE2297 and with pKR1333 (SEQID NO:266), having experiment number MSE2298. Control vector pKR1256(SEQ ID NO:98; Example 16), comprising Yarrowia DGAT2, was alsotransformed alone in a similar way for a control and has experimentnumber MSE2295. Events were selected and somatic embryos matured in SHaMas described in Example 5. Oil concentrations and fatty acid profileswere determined as described in Example 5 for MSE2295, MSE2296, MSE 2297and MSE2298 and results for each experiment are shown in Table 54, Table55, Table 56 and Table 57, respectively.

TABLE 54 Oil concentrations and fatty acid profiles for events fromMSE2295 MSE2295 (YL DGAT2) Event 16:0 18:0 18:1 18:2 18:3 % Oil 2295-712.3 5.5 37.0 38.1 7.1 13.4 2295-26 11.9 5.7 42.1 33.6 6.7 12.8 2295-414.0 5.1 29.0 41.5 10.4 11.4 2295-15 13.0 6.5 34.0 37.3 9.1 10.0 2295-213.9 6.2 32.6 37.6 9.7 9.7 2295-14 14.1 5.1 28.5 40.6 11.7 8.8 2295-1015.9 6.1 27.8 39.1 11.2 8.6 2295-8 13.9 5.9 32.4 37.7 10.2 8.2 2295-517.1 5.7 21.3 42.8 13.1 8.0 2295-1 16.6 4.4 17.3 47.9 13.8 7.9 2295-1814.3 5.5 34.0 35.5 10.8 7.6 2295-24 15.5 4.8 23.6 42.7 13.4 7.2 2295-2317.0 4.9 17.4 44.9 16.0 6.6 2295-17 15.6 6.8 30.5 35.4 11.7 6.6 2295-2014.1 5.6 27.6 39.2 13.5 6.5 2295-25 16.3 5.2 20.4 41.9 16.0 6.3 2295-1314.7 5.6 23.5 40.9 15.3 6.3 2295-16 16.9 7.2 24.4 38.4 13.1 6.2 2295-618.4 6.1 20.9 40.0 14.6 6.1 2295-21 18.1 5.2 15.1 45.5 16.1 5.9 2295-2216.4 5.1 21.2 41.7 15.6 5.9 2295-3 17.9 5.5 21.3 40.3 14.9 5.8 2295-1914.2 5.0 19.9 45.1 15.9 5.6 2295-9 16.3 4.5 23.4 40.9 14.9 5.2 2295-1116.6 6.1 22.4 39.1 15.8 4.5 2295-12 18.3 5.0 16.3 41.7 18.7 4.3 Avg.15.5 5.6 25.5 40.4 13.0 7.5

TABLE 55 Oil concentrations and fatty acid profiles for events fromMSE2296 MSE2296 (MA DGAT2) Event 16:0 18:0 18:1 18:2 18:3 % Oil 2296-2613.4 5.3 31.5 41.8 8.0 13.4 2296-14 13.4 5.9 32.6 38.9 9.2 11.6 2296-2313.9 6.0 32.5 39.5 8.1 11.0 2296-4 15.5 5.4 25.4 43.1 10.6 10.9 2296-1715.4 5.2 18.9 49.1 11.4 10.8 2296-6 14.5 6.7 31.9 37.5 9.3 10.3 2296-2414.3 6.1 29.6 40.5 9.5 10.1 2296-16 14.6 5.8 30.0 39.3 10.3 10.0 2296-1915.5 6.3 25.6 40.5 12.0 9.4 2296-12 15.1 6.0 28.6 39.8 10.6 8.6 2296-2216.4 4.4 17.1 47.9 14.3 8.5 2296-20 14.9 6.6 27.2 39.9 11.5 8.3 2296-1316.4 5.5 20.2 44.6 13.3 8.2 2296-11 16.0 6.0 22.8 42.0 13.2 8.2 2296-315.2 5.6 22.8 44.4 12.0 8.2 2296-1 16.2 4.7 20.5 45.6 13.2 7.8 2296-1814.9 5.1 28.2 39.4 12.3 7.5 2296-9 15.9 7.8 21.1 42.5 12.6 7.4 2296-1517.1 5.1 18.8 44.3 14.7 7.4 2296-10 16.5 6.7 25.0 39.7 12.2 7.3 2296-514.2 6.7 29.1 38.3 11.7 7.2 2296-25 16.5 6.1 22.1 41.4 13.8 7.0 2296-2116.4 5.8 23.6 41.2 12.9 6.4 2296-8 16.8 7.1 21.7 41.5 12.9 6.3 2296-2716.5 4.6 17.5 44.1 17.4 5.8 2296-7 17.6 5.9 21.6 38.1 16.8 4.9 2296-214.3 5.4 19.4 41.3 19.6 3.5 Avg. 15.5 5.9 24.6 41.7 12.3 8.4

TABLE 56 Oil concentrations and fatty acid profiles for events fromMSE2297 MSE2297 (PA DGAT2) Event 16:0 18:0 18:1 18:2 18:3 % Oil 2297-2317.0 5.8 22.1 42.1 12.9 7.2 2297-10 16.5 6.3 21.6 42.2 13.3 7.2 2297-2116.5 5.1 17.1 46.9 14.4 7.0 2297-1 16.4 6.7 22.9 40.8 13.2 6.7 2297-1517.2 5.4 18.8 44.5 14.0 6.4 2297-22 19.6 5.0 13.5 37.8 24.1 6.2 2297-1817.3 6.9 21.9 40.3 13.6 6.0 2297-17 16.2 6.6 23.7 39.7 13.7 5.9 2297-516.8 5.5 18.6 43.7 15.4 5.9 2297-3 17.4 4.8 18.9 42.3 16.6 5.7 2297-617.0 6.1 18.8 41.9 16.2 5.6 2297-13 17.1 6.0 22.5 39.0 15.3 5.5 2297-1616.9 5.8 21.0 42.3 14.0 5.5 2297-11 17.1 5.2 21.1 41.2 15.4 5.4 2297-2017.0 5.9 21.9 39.5 15.6 5.4 2297-25 15.8 4.6 22.0 41.1 16.5 5.3 2297-2417.0 5.6 21.7 42.5 13.1 5.2 2297-9 17.3 5.3 17.9 41.3 18.2 5.1 2297-1416.7 5.4 19.6 42.6 15.7 5.1 2297-7 17.0 5.0 20.7 42.0 15.3 4.8 2297-415.9 5.1 19.7 40.8 18.6 4.6 2297-12 18.7 5.3 18.7 40.3 17.0 4.5 2297-2618.5 4.8 16.8 41.1 18.7 4.3 2297-8 17.5 5.2 18.6 39.5 19.2 4.2 2297-1918.0 5.0 18.0 40.6 18.4 4.2 2297-2 18.5 5.9 18.3 38.2 19.0 3.5 Avg. 17.25.6 19.9 41.3 16.1 5.5

TABLE 57 Oil concentrations and fatty acid profiles for events fromMSE2298 MSE2298 (RG DGAT2) Event 16:0 18:0 18:1 18:2 18:3 % Oil 2298-814.7 5.0 22.6 44.4 13.2 9.4 2298-16 17.1 5.4 26.2 41.1 10.3 9.4 2298-1116.5 4.8 21.0 45.0 12.7 9.2 2298-21 15.8 5.4 25.0 41.5 12.3 8.5 2298-2416.4 4.8 20.9 44.1 13.7 8.2 2298-5 16.4 6.0 26.9 39.5 11.2 8.2 2298-418.3 6.5 24.2 38.8 12.2 7.9 2298-22 16.5 5.6 23.2 41.5 13.1 7.6 2298-216.6 5.8 27.1 39.2 11.4 7.4 2298-14 17.2 5.9 28.0 36.3 12.6 7.4 2298-1816.8 6.0 24.4 39.8 13.0 7.3 2298-19 17.2 6.3 30.0 35.3 11.2 7.2 2298-2317.0 6.7 25.8 38.3 12.2 6.8 2298-10 15.2 4.5 22.4 41.6 16.4 6.7 2298-316.9 6.5 23.3 38.9 14.3 6.3 2298-6 17.2 6.0 24.3 38.4 14.0 6.0 2298-716.3 5.1 21.9 40.4 16.3 6.0 2298-17 16.5 5.3 17.5 41.7 19.0 5.9 2298-2016.6 5.9 23.2 39.5 14.8 5.5 2298-9 17.1 5.2 19.5 41.7 16.5 5.5 2298-1217.8 5.1 20.4 39.2 17.5 5.2 2298-13 15.7 5.1 20.4 39.8 18.9 5.1 2298-117.6 4.8 19.0 41.6 17.0 4.9 2298-15 16.7 6.1 23.3 38.2 15.7 4.4 Avg.16.7 5.6 23.4 40.3 14.1 6.9

MA_DGAT2 increases oil and oleic acid concentration in somatic embryosto amounts similar to YL DGAT2. MA_DGAT2 is also similar to YL_DGAT2 inthat palmitic acid and alpha-linolenic acid concentrations aredecreased. RG DGAT2 has some ability to increase oil and oleic acidconcentrations in somatic embryos but the overall effect is less thanfor either MA DGAT2 or YL DGAT2. PA DGAT2 does not increase oil or oleicacid concentrations in this experiment.

Example 29 Expression of DGAT2s from Yarrowia lipolyitica and Lipomycesstarkeyi in Soybean Somatic Embryos

The present example describes construction of pKR1337, comprisingLipomyces starkeyi diacylglyceride acyltransferase 2 (LS_DGAT2) andexpression in somatic embryos. Control vector pKR1256 (SEQ ID NO:98;Example 16), comprising Yarrowia DGAT2, was also expressed forcomparison.

Construction of pKR1337, comprising LS DGAT2

Because the LS DGAT2 genomic sequence (SEQ ID NO:194; Example 22)appeared to contain numerous introns when compared to other DGATs byBlastX analysis, the LS DGAT2 coding sequence was cloned by PCR fromcDNA. RNA isolation and cDNA synthesis is described below.

RNA was isolated from Lipomyces starkeyi and cDNA was synthesized asdescribed in Example 22. Analysis of the LS DGAT2 genomic sequence (SEQID NO:194; Example 22) suggested two potential ORF initiation sites and5′ oligos were constructed to each of these potential ORF initiationsites. Based on the genomic DNA sequence (SEQ ID NO:194; Example 22) andusing the first potential initiation site (longer ORF), LS DGAT2 wasamplified from the cDNA (2 μL) with oligonucleotide primers Lip5-1 (SEQID NO:267) and Lip3 (SEQ ID NO:268), using the Phusion™ High-FidelityDNA Polymerase (Cat. No. F553S, Finnzymes Oy, Finland) following themanufacturer's protocol. Using the second potential initiation site(slightly shorter ORF), LS DGAT2 was amplified from the cDNA (2 μL) witholigonucleotide primers LipS-2 (SEQ ID NO:269) and Lip3 (SEQ ID NO:268)in the same way. Only the PCR reaction using the second potentialinitiation site (Lip5-2 with Lip3) gave a PCR product. The resulting DNAfragment was cloned into the pCR-Blunt® cloning vector using the ZeroBlunt® PCR Cloning Kit (Invitrogen Corporation), following themanufacturer's protocol, to produce pHD30 (SEQ ID NO: 270). The LS DGAT2cDNA nucleotide and corresponding amino acid sequences are set forth inSEQ ID NO:271 and SEQ ID NO:272, respectively. A BLASTP search of theannotated fungal protein database at NCBI was conducted. Based on aCLUSTALW alignment, the deduced amino acid sequence of the LS DGAT2 cDNAshares 51.0% sequence identity to a hypothetical protein product fromYarrowia lipolytica CLIB122 with GENBANK accession number X1³ _(—)504700which represents the protein with the closest similarity in thisdatabase. The amino acid sequence of this protein identical to that ofYL DGAT 2 described in PCT Publication No. WO 2005/003322 and differs inone amino acid residue from that of SEQ ID NO:10 for reasons that areoutline in Example 1.

The NotI fragment of pHD30 (SEQ ID NO:270), containing LS_DGAT2, wascloned into the NotI site of pKR72 (SEQ ID NO:26; Example 4) to producepKR1337 (SEQ ID NO:273).

Expression of LS DGAT2 in Soy Somatic Embryos

Soybean embryogenic suspension culture (cv. Jack) was transformed withpKR1337 (SEQ ID NO:273), having experiment number MSE2333. Controlvector pKR1256 (SEQ ID NO:98; Example 16), comprising Yarrowia DGAT2,was also transformed alone in a similar way for a control and hasexperiment number MSE2334.

Example 30 Expression of DGAT1s from Yarrowia lipolytica and Mortierellaalpina in Soybean Somatic Embryos

The present example describes construction of pKR1334, comprisingMortierella diacylglyceride acyltransferase 1 (Ma_DGAT1) and controlvector pKR1323, comprising Yarrowia DGAT1 codon optimized for expressionin soy.

Construction of pKR1334, comprising MA_DGAT1

MA_DGAT1 was amplified from pMDGAT1-17 (SEQ ID NO:274), which isdescribed in issued U.S. Pat. No. 7,273,746 (the contents of which isincorporated by reference) with oligonucleotide primers oMaDG1-1 (SEQ IDNO:275) and oMaDG1-2 (SEQ ID NO:276), using the Phusion™ High-FidelityDNA Polymerase (Cat. No. F553S, Finnzymes Oy, Finland) following themanufacturer's protocol. The resulting DNA fragment was cloned into thepCR-Blunt® cloning vector using the Zero Blunt® PCR Cloning Kit(Invitrogen Corporation), following the manufacturer's protocol, toproduce pKR1329 (SEQ ID NO:277). The MA_DGAT1 nucleotide andcorresponding amino acid sequences are set forth in SEQ ID NO:278 andSEQ ID NO:279, respectively.

The NotI fragment of pKR1329 (SEQ ID NO:277), containing MA_DGAT1, wascloned into the NotI site of pKR72 (SEQ ID NO:26; Example 4) to producepKR1334 (SEQ ID NO:280).

Construction of pKR1323, comprising YL DGAT1 Codon Optimized for Soy

YL DGAT1 was codon optimized for expression in soy as described inExample 9 and Example 17 and the codon optimized nucleotide sequence isset forth in SEQ ID NO:64. The corresponding amino acid sequence codedfor by the codon optimized YL DGAT 1 is identical to the wild-type YLDGAT1 amino acid sequence (SEQ ID NO:65). The codon optimized sequencecontained two internal ORF of 1023 bp and 687 bp in length in theopposite direction as the coding sequence was and these were not presentin the wild-type YL_DGAT1 sequence. In order to remove the internalORFs, the following cloning steps were carried out.

In order to add appropriate restriction sites, the NotI fragment ofKS392 (SEQ ID NO:101, Example 17), containing the codon optimized YLDGAT1, was cloned into the NotI site pBluescript II SK(+) vector(Stratagene), to produce pKR1314 (SEQ ID NO:281).

In order to remove the internal 687 bp ORF, the 5′ end of YL DGAT1 wasamplified from KS392 (SEQ ID NO:101) with oligonucleotide primersoY1DG1co-1 (SEQ ID NO:282) and oY1DG1co-2 (SEQ ID NO:283), using thePhusion™ High-Fidelity DNA Polymerase (Cat. No. F553S, Finnzymes Oy,Finland) following the manufacturer's protocol. The 5′ end of YL DGAT1was also amplified from KS392 (SEQ ID NO:101) with oligonucleotideprimers oY1DG1co-3 (SEQ ID NO:284), which is complementary to oYLDG1co-2(SEQ ID NO:283) and oY1DG1co-4 (SEQ ID NO:285), which removes theinternal 1023 bp ORF, using the Phusion™ High-Fidelity DNA Polymerase(Cat. No. F553S, Finnzymes Oy, Finland) following the manufacturer'sprotocol. YL DGAT1 was then amplified from the combined PCR products ofthe two PCR reactions described above with oligonucleotide primersoY1DG1co-1 (SEQ ID NO:282) and oY1DG1co-4 (SEQ ID NO:284), using thePhusion™ High-Fidelity DNA Polymerase (Cat. No. F553S, Finnzymes Oy,Finland) following the manufacturer's protocol. The resulting DNAfragment was cloned into the pCR-Blunt® cloning vector using the ZeroBlunt® PCR Cloning Kit (Invitrogen Corporation), following themanufacturer's protocol, to produce pKR1310 (SEQ ID NO:286).

The BglII/EcoRV fragment of pKR1314 (SEQ ID NO:281) containing theremaining 3′ end of the YL-DGAT 1 gene was cloned into the BglII/EcoRVfragment of pKR1310 (SEQ ID NO:286) to produce pKR1316 (SEQ ID NO:287),which contains a full length, codon optimized YL DGAT1 with all internalORFs removed. The nucleotide sequence for the codon optimized YL DGAT1having internal ORFs removed, called YL DGAT1cod2, is set forth in SEQID NO:288. The amino acid sequence of YL DGAT1cod2 is identical to thewild-type YL DGAT1 (SEQ ID NO:65).

The NotI fragment of pKR1316 (SEQ ID NO:287), containing YL DGAT1cod2,was cloned into the NotI site of pKR72 (SEQ ID NO:26; Example 4) toproduce pKR1323 (SEQ ID NO:289).

Expression of YL DGAT1cod2 and Ma DGAT1 in Soy Somatic Embryos

Soybean embryogenic suspension culture (cv. Jack) was transformed withpKR1334 (SEQ ID NO:280), having experiment number MSE2332. Controlvector pKR1323 (SEQ ID NO:289), comprising YL DGAT1cod2, was alsotransformed in a similar way and has experiment number MSE2331.

Example 31 Expression of DGAT2s from Yarrowia lipolyitica and Phaffiarhodozyma in Soybean Somatic Embryos

The present example describes construction of pKR1372, comprisingPhaffia rhodozyma diacylglyceride acyltransferase 2 (PR DGAT2) andexpression in somatic embryos. Control vector pKR1256 (SEQ ID NO:98;Example 16), comprising Yarrowia DGAT2, was also expressed forcomparison.

Construction of pKR1372, comprising PR DGAT2

Based on the cDNA sequence of PR_DGAT2 RACE (SEQ ID NO:214; Example 24)PR DGAT2 was amplified from the cDNA (2 μL) with oligonucleotide primersPrDGAT2-5 (SEQ ID NO:290) and PrDGAT2-3 (SEQ ID NO:291), using thePhusion™ High-Fidelity DNA Polymerase (Cat. No. F553S, Finnzymes Oy,Finland) following the manufacturer's protocol, to produce pHD37 (SEQ IDNO:292). The insert of pHD37 comprising the DGAT2 gene was sequencedcompletely. The resulting DNA sequence is referred to as PR_DGAT2. It'snucleotide sequences is set forth as SEQ ID NO:293. There are eightdifferences at the nucleotide level between PR_DGAT2 RACE (SEQ IDNO:214) and PR_DGAT2 (SEQ ID NO:293) nucleotide sequences however theamino acid sequences that can be deduced from both sequences areidentical. The amino acid sequence of PR_DGAT2 is set forth as (SEQ IDNO:215).

The NotI fragment of pHD37 (SEQ ID NO:292), containing PR_DGAT2, wascloned into the NotI site of pKR₇₂ (SEQ ID NO:26; Example 4) to producepKR1372 (SEQ ID NO:294).

Expression of PR DGAT2 in Soy Somatic Embryos

Soybean embryogenic suspension culture (cv. Jack) was transformed withpKR1372 (SEQ ID NO:294), having experiment number MSE2351. Controlvector pKR1256 (SEQ ID NO:98; Example 16), comprising Yarrowia DGAT2,was also transformed alone in a similar way for a control and hasexperiment number MSE2349.

Example 32 Expression of DGAT1s from Yarrowia lipolytica and Lipomycesstarkeyi in Soybean Somatic Embryos

The present example describes construction of pKR1375, comprisingLipomyces diacylglyceride acyltransferase 1 (LS DGAT1) and expression insomatic embryos. Control vector pKR1323 (SEQ ID NO:289) described inExample 30, comprising YL DGAT1cod2, was also expressed for comparison.

Construction of pKR1375, comprising LS DGAT1

Because the LS_DGAT1 genomic sequence (SEQ ID NO:189; Example 22)appeared to contain numerous introns when compared to other DGATs byBlastX analysis, the LS_DGAT1 coding sequence was cloned by PCR fromcDNA. cDNA synthesis is described in Example 22.

Based on the genomic sequence of LS_DGAT1 (SEQ ID NO:189). LS_DGAT1 wasamplified from cDNA with oligonucleotide primers oLsDGAT1-5-1 (SEQ IDNO:295) and oLsDGAT1-3 (SEQ ID NO:296), using the Phusion™ High-FidelityDNA Polymerase (Cat. No. F553S, Finnzymes Oy, Finland) following themanufacturer's protocol. Isolation of RNA and synthesis of cDNA fromLipomyces starkeyi is described in Example 22. The resulting DNAfragment was cloned into the pCR-Blunt® cloning vector using the ZeroBlunt® PCR Cloning Kit (Invitrogen Corporation), following themanufacturer's protocol, to produce pHD38 (SEQ ID NO:297). Nucleotideand corresponding amino acid sequences of the Lipomyces starkeyi DGAT1transcript are set forth in SEQ ID NO:298 and SEQ ID NO:299,respectively. A BLASTP search of the annotated fungal protein databaseat NCBI was conducted. The deduced amino acid sequence of the Lipomycesstarkeyi DGAT1 transcript shares 52.4% sequence identity to ahypothetical protein product from Coccidioides immitis with GENBANKaccession number XP_(—)001247089 which represents the protein with theclosest similarity in this database. The protein has been annotated asmember of the MBOAT (membrane bound O-acyl transferase) family ofmembrane proteins that contains a variety of acyltransferase enzymes.

The NotI fragment of pHD38 (SEQ ID NO:297), containing LS_DGAT1, wascloned into the NotI site of pKR72 (SEQ ID NO:26; Example 4) to producepKR1375 (SEQ ID NO:300).

Expression of YL DGAT1cod2 and LS DGAT1 in Soy Somatic Embryos

Soybean embryogenic suspension culture (cv. Jack) was transformed withpKR1375 (SEQ ID NO:300), having experiment number MSE2352. Controlvector pKR1323 (SEQ ID NO:289), comprising YL DGAT1cod2, was alsotransformed in a similar way and has experiment number MSE2350.

Example 33 DGAT 1 Gene Sequences from Mortierella alpina and Lipomycesstarkeyi that are Optimized for Expression in Soy Beans

Nucleotide sequences encoding MA DGAT1 (SEQ ID NO:278) and LS DGAT1 (SEQID NO:298) were re-designed for optimized expression in soybean seedusing methods similar to those described in Wu, G et al. Nucleic AcidsResearch (2007), 35: D76-D79; Villalobos, A. et al. BMC Bioinformatics(2006), 7 No pp. given; Wu, G. et al. Protein Expression andPurification (2006), 47: 441-445; Richardson, S. M. et al. GenomeResearch (2006), 16: 550-556; Jayaraj, S. et al. Nucleic Acids Research(2005) 33: 3011-3016. DNA molecules were synthesized by Codon Devices(MA, USA). Expression-optimized DNA sequences of MA DGAT1 and LS DGAT1are set forth in SEQ ID NO:301 and SEQ ID NO:302, respectively. Theamino acid sequences of proteins encoded by these DNA sequences at setforth as SEQ ID NO:279 and SEQ ID NO:299.

Example 34 Expression Optimized DGAT2 Gene Sequences Encoding DGAT2Protein Variants

Nucleotide sequences encoding DGAT2 proteins from Torulasporadelbrueckii, Pichia anomala, Debaryomyces hansenii, Candida zeylanoides,Lipomyces starkeyi, Mucor circinelloides, Phaffia rhodozyma, Rhodotorulaglutinis and Mortierella alpina described herein (see previous Examples18-32) were re-designed for optimized expression in soybean seed usingmethods similar to those described in Wu, G et al. Nucleic AcidsResearch (2007), 35: D76-D79; Villalobos, A. et al. BMC Bioinformatics(2006), 7 No pp. given; Wu, G. et al. Protein Expression andPurification (2006), 47: 441-445; Richardson, S. M. et al. GenomeResearch (2006), 16: 550-556; Jayaraj, S. et al. Nucleic Acids Research(2005) 33: 3011-3016. DNA molecules were synthesized by Codon Devices(MA, USA). Expression-optimized DNA sequences and DGAT2 proteins encodedby said DNA sequences and related SEQ ID NOs are shown in Table 58. AllDGAT2 variants contain the motif FxxPxFR that was altered from FxxPxYRpresent in the native DGAT2 proteins.

TABLE 58 Gene name ORF (SEQ ID NO:) AA (SEQ ID NO:) TD_DGAT2Acod 303 304TD_DGAT2Bcod 305 306 PA_DGAT2cod 307 308 DH_DGAT2cod 309 310 CZ_DGAT2cod311 312 LS_DGAT2cod 313 314 MC_DGAT2cod 315 316 PR_DGAT2cod 317 318RG_DGAT2cod 319 320 MA_DGAT2cod 321 322

Example 35 Co-Expression of YL DGAT1 with a FAD2/TE2 Down RegulationConstruct in Soybean Somatic Embryos

The present example describes construction of soybean expression vectorspKR1274, comprising Yarrowia DGAT1 (YL DGAT1) and either pKR1267 orpKR1269, comprising a soybean fatty acid desaturase 2 (GMFAD2)/thioesterase 2 (GM TE2) down-regulation construct. While the GMFAD2-TE2 down-regulation region of pKR1267 and pKR1269 are identical ineach construct and both are driven by the KTi3 promoter, pKR1267contains only the KTi3 terminator and pKR1269 contains both the KTi3 andsoy albumin terminators.

Construction of pKR1274 comprising YL DGAT 1

A starting plasmid pKR85 (SEQ ID NO:27), which was previously describedin Example 4 contains the hygromycin B phosphotransferase gene (HPT)(Gritz, L. and Davies, J., Gene 25:179-188 (1983)), flanked by the T7promoter and transcription terminator (T7prom/hpt/T7term cassette), anda bacterial origin of replication (ori) for selection and replication inbacteria (e.g., E. coli). In addition, pKR72 also contains thehygromycin B phosphotransferase gene, flanked by the 35S promoter (Odellet al., Nature 313:810-812 (1985)) and NOS 3′ transcription terminator(Depicker et al., J. Mol. Appl. Genet. 1:561-570 (1982)) (35S/hpt/NOS3′cassette) for selection in plants such as soybean. Plasmid pKR85 (SEQ IDNO:27) also contains a NotI restriction site, flanked by the promoterfor the α′ subunit of β-conglycinin (Beachy et al., EMBO J. 4:3047-3053(1985)) and the 3′ transcription termination region of the phaseolingene (Doyle et al., J. Biol. Chem. 261:9228-9238 (1986)), calledBcon/NotI/Phas3′ cassette.

The Bcon/NotI/Phas3′ cassette was removed from pKR85 (SEQ ID NO:27) bydigestion with HindIII and the resulting fragment was re-ligated toproduce pKR278 (SEQ ID NO:323).

The BsiWI fragment of pKR1235 (SEQ ID NO:74, Example 14), containing theYL DGAT1, was cloned into the BsiWI site of pKR278 (SEQ ID NO:323),which was previously described in US Pat. Pub. US20080095915 (thecontents of which are incorporated by reference), to produce pKR1274(SEQ ID NO:324).

Construction of pKR1267 comprising GM FAD2-TE2 Down-Regulation Cassette

The 5′ end of GM TE2 (SEQ ID NO:325) was amplified from pTC4 (SEQ IDNO:326), which was previously described in WO1996006936A1 (the contentsof which are incorporated by reference), with oligonucleotide primersGmTE2_(—)5-1 (SEQ ID NO:327) and GmTE2_(—)3-1 (SEQ ID NO:328), using thePhusion™ High-Fidelity DNA Polymerase (Cat. No. F553S, Finnzymes Oy,Finland) following the manufacturer's protocol. The 3′ end of GM TE2(SEQ ID NO:325) was amplified from pTC4 (SEQ ID NO:326) witholigonucleotide primers GmTE2_(—)5-2 (SEQ ID NO:329) and GmTE2_(—)3-2(SEQ ID NO:330), using the Phusion™ High-Fidelity DNA Polymerase (Cat.No. F553S, Finnzymes Oy, Finland) following the manufacturer's protocol.The resulting two PCR products were combined and amplified withGmTE2_(—)5-1 (SEQ ID NO:327) and GmTE2_(—)3-2 (SEQ ID NO:330) using thePhusion™ High-Fidelity DNA Polymerase (Cat. No. F553S, Finnzymes Oy,Finland) following the manufacturer's protocol. The resulting DNAfragment was cloned into the pCR-Blunt® cloning vector using the ZeroBlunt® PCR Cloning Kit (Invitrogen Corporation), following themanufacturer's protocol, to produce pKR1258 (SEQ ID NO:331).

The 5′ end of GM FAD2 (SEQ ID NO:332) was amplified from pBS43 (SEQ IDNO:333), which was previously described in WO1997047731A2 (the contentsof which are incorporated by reference), with oligonucleotide primersGmFAD2-1_(—)5-1 (SEQ ID NO:334) and GmFAD2-1_(—)3-1 (SEQ ID NO:335),using the Phusion™ High-Fidelity DNA Polymerase (Cat. No. F553S,Finnzymes Oy, Finland) following the manufacturer's protocol. The 3′ endof GM FAD2-1 (SEQ ID NO:332) was amplified from pBS43 (SEQ ID NO:333)with oligonucleotide primers GmFAD2-1_(—)5-2 (SEQ ID NO:336) andGmFAD2-1_(—)3-2 (SEQ ID NO:337), using the Phusion™ High-Fidelity DNAPolymerase (Cat. No. F553S, Finnzymes Oy, Finland) following themanufacturer's protocol. The resulting two PCR products were combinedand amplified with GmFAD2-1_(—)5-1 (SEQ ID NO:334) and GmFAD2-1_(—)3-2(SEQ ID NO:337) using the Phusion™ High-Fidelity DNA Polymerase (Cat.No. F553S, Finnzymes Oy, Finland) following the manufacturer's protocol.The resulting DNA fragment was cloned into the pCR-Blunt® cloning vectorusing the Zero Blunt® PCR Cloning Kit (Invitrogen Corporation),following the manufacturer's protocol, to produce PCRblunt-Fad2-1 (SEQID NO:338).

The MluI fragment of pKR1258 (SEQ ID NO:331), containing GM TE2, wascloned into the MluI fragment of PCRblunt-Fad2-1 (SEQ ID NO:338),containing GM FAD2-1, to produce pKR1259 (SEQ ID NO:339).

The EcoRI fragment of pKR1259 (SEQ ID NO:339) which comprises the 5′ endof the GM FAD2/TE2 fragment, was cloned into the MfeI site of pKR1259(SEQ ID NO:339) to produce pKR1261 (SEQ ID NO:340). This DNA constructcontains a GM FAD2-TE2-TE2loop-TE2-FAD2 haripin structure flanked byNotI sites.

The NotI fragment of pKR1261(SEQ ID NO:340), containing GMFAD2-TE2-TE2loop-TE2-FAD2, was cloned into the NotI site of pKR123R (SEQID NO:341), which was previously described in WO2004071467A2 (thecontents of which are incorporated by reference), to produce pKR1266(SEQ ID NO:342).

The BsiWI/PstI fragment of pKR1266 (SEQ ID NO:342), containing the GMFAD2-TE2-TE2loop-TE2-FAD2 was cloned into the BsiWI/SbfI fragment ofpKR278 (SEQ ID NO:323) to produce pKR1267 (SEQ ID NO:343).

Construction of pKR1267 comprising GM FAD2-TE2 Down-Regulation Cassette

The NotI fragment of pKR1261(SEQ ID NO:340), containing GMFAD2-TE2-TE2loop-TE2-FAD2, was cloned into the NotI site of pKR457 (SEQID NO:344), which was previously described in PCT Publication No. WO2005/047479 (the contents of which are hereby incorporated byreference), to produce pKR1264 (SEQ ID NO:345).

The PstI fragment of pKR1264 (SEQ ID NO:345), containing the GMFAD2-TE2-TE2loop-TE2-FAD2 was cloned into the SbfI fragment of pKR277(SEQ ID NO:346), which was previously described in PCT Publication No.WO 2004/071467 to produce pKR1269 (SEQ ID NO:347).

Co-expression of GM FAD2-TE2-TE2loop-TE2-FAD2 Down-Regulation ConstructsEither alone or with YL DGAT2

Soybean embryogenic suspension culture (cv. Jack) was transformed withthe pKR1267 (SEQ ID NO:343) alone and having experiment number MSE2213or with the BsiWI fragment of pKR1269 (SEQ ID NO:347) and pKR1274 (SEQID NO:324) and having experiment number MSE2210. Events were selectedand somatic embryos matured in SHaM as described in Example 5. Oilconcentrations and fatty acid profiles were determined as described inExample 4 for MSE2213 and MSE2210 and results for each experiment areshown in Table 59 and Table 60, respectively.

TABLE 59 Oil concentrations and fatty acid profiles for events fromMSE2213 MSE2213 (GM FAD2-TE2-TE2loop-TE2-FAD2) Event 16:0 18:0 18:1 18:218:3 % Oil 2213-16 5.8 2.6 72.7 10.5 8.5 16.8 2213-26 12.0 5.7 24.8 47.210.4 16.5 2213-30 11.7 5.3 18.5 53.1 11.4 15.4 2213-23 9.0 3.2 52.2 24.711.0 15.3 2213-29 13.1 3.7 13.3 57.1 12.8 15.0 2213-7 6.4 3.1 66.3 15.68.6 14.9 2213-31 11.7 3.0 26.9 46.5 11.9 14.5 2213-13 6.7 3.3 64.5 16.68.9 14.4 2213-5 3.5 3.0 78.0 7.5 8.1 13.8 2213-9 7.6 3.4 57.6 20.3 11.013.2 2213-20 7.8 4.1 56.7 20.8 10.6 13.1 2213-4 4.1 2.7 72.1 11.1 10.012.7 2213-17 7.7 5.2 57.0 19.7 10.4 12.5 2213-3 7.4 3.6 64.4 14.4 10.212.5 2213-24 12.9 7.3 29.4 39.0 11.4 12.1 2213-1 13.8 7.1 22.1 44.6 12.411.9 2213-6 6.7 2.2 57.2 22.2 11.7 11.8 2213-11 9.2 5.2 58.2 16.4 11.010.9 2213-2 7.8 4.3 45.3 31.0 11.7 10.6 2213-14 7.3 4.6 63.0 15.0 10.210.5 2213-27 8.5 6.0 48.9 25.0 11.6 10.1 2213-19 8.0 4.0 53.7 21.4 12.89.9 2213-21 11.1 5.6 28.3 40.6 14.4 9.8 2213-18 7.4 4.1 57.2 17.7 13.59.4 2213-12 6.4 4.3 63.2 15.5 10.6 9.4 2213-10 13.8 7.0 24.0 42.1 13.29.4 2213-28 8.8 4.1 54.1 19.1 13.8 9.1 2213-25 6.9 3.5 53.9 21.9 13.89.0 2213-15 5.2 3.4 61.7 17.6 12.1 8.0 2213-8 14.4 6.6 20.0 42.9 16.17.6 2213-22 14.3 6.3 21.3 41.9 16.2 7.2 Avg. 8.9 4.4 47.9 27.1 11.6 11.9

TABLE 60 Oil concentrations and fatty acid profiles for events fromMSE2210 MSE2210 (YL DGAT1 & GM FAD2-TE2-TE2loop-TE2-FAD2) Event 16:018:0 18:1 18:2 18:3 % Oil 2210-2 3.8 2.7 74.7 13.1 5.8 22.1 2210-23 10.36.8 32.0 44.4 6.5 20.0 2210-29 7.5 5.5 63.9 17.1 6.0 20.0 2210-19 3.93.5 79.1 8.1 5.4 19.7 2210-10 7.3 3.8 66.6 15.6 6.7 19.6 2210-12 5.7 3.667.6 17.6 5.6 19.6 2210-14 4.5 2.4 67.9 17.8 7.4 19.3 2210-25 5.1 3.571.8 13.1 6.4 18.4 2210-5 9.0 4.1 43.1 35.9 7.9 17.8 2210-24 13.7 4.320.5 52.5 9.1 17.5 2210-13 10.2 5.5 36.8 40.4 7.0 17.4 2210-11 10.9 7.830.6 43.2 7.5 16.6 2210-1 4.1 3.0 75.6 9.6 7.7 15.9 2210-6 2.7 1.7 83.95.3 6.4 15.5 2210-16 8.4 3.7 48.5 31.3 8.1 15.3 2210-26 7.1 4.9 55.224.7 8.1 14.4 2210-7 4.3 3.2 62.9 20.8 8.7 14.3 2210-20 6.8 4.3 65.215.7 7.9 13.8 2210-27 10.9 7.4 42.4 30.0 9.3 13.8 2210-30 5.0 2.4 65.117.1 10.4 13.7 2210-3 7.1 4.8 52.9 27.4 7.9 13.6 2210-4 6.0 3.7 66.015.9 8.5 13.3 2210-22 3.2 3.3 77.7 8.1 7.8 13.2 2210-8 9.1 4.9 49.7 27.39.0 13.1 2210-21 6.0 3.5 67.8 14.9 7.8 12.8 2210-18 12.7 5.2 20.2 49.712.1 12.5 2210-31 4.4 2.9 73.1 10.1 9.5 11.7 2210-9 4.0 3.1 74.1 9.3 9.611.2 2210-15 3.5 2.4 72.5 11.0 10.6 11.1 2210-28 14.1 5.7 20.2 45.5 14.510.1 2217-29 12.0 7.5 28.0 38.8 13.8 8.7 Avg. 7.2 4.2 56.6 23.6 8.4 15.4

Comparison of results in Tables 59 and 60 demonstrates that combinationof YL DGAT1 expression with down-regulation of GM FAD2-1 and GM TE2changes the fatty acid profile to an extend that exceeds the change thatobserved when only FAD2-1 and TE2 genes are suppressed.

Example 36 Cloning the cDNA for DGAT2 from Cryptococcus curvatus

Because the CC DGAT2 genomic sequence (SEQ ID NO:226; Example 25)appeared to contain numerous introns when compared to other DGATs byBlastX analysis, the CC DGAT2 coding sequence was cloned by PCR fromcDNA.

RNA was isolated from Cryptococcus curvatus and cDNA was synthesized asdescribed in Example 25. Analysis of the CC DGAT2 genomic sequence (SEQID NO:226; Example 25) suggested a potential ORF initiation site and a5′ oligo was constructed to the ORF initiation site. CC DGAT2 wasamplified from the cDNA (2 μl) with oligonucleotide primers CC ORF FWD(SEQ ID NO:348) and CC ORF REV (SEQ ID NO:349), using GoTaq polymerase(Promega, USA) following the manufacturer's protocol. The resulting DNAfragment was cloned into pGEM T-easy (Promega) using manufacturerinstructions and sequenced. The CC DGAT2 cDNA nucleotide andcorresponding amino acid sequences are set forth in SEQ ID NO:350 andSEQ ID NO:351, respectively. A BLASTP search of the annotated fungalprotein database at NCBI was conducted. Based on a CLUSTALW alignment,the deduced amino acid sequence of the CC DGAT2 cDNA shares 61% sequenceidentity to a DGAT2 protein product from Cryptococcus neoformans withGENBANK accession number XP_(—)571236 which represents the protein withthe closest similarity in this database.

Example 37 Functional Analysis of DGAT2s from Yarrowia lipolyitica andLipomyces starkeyi and DGAT1s from Yarrowia lipolytica and Mortierellaalpina in Soybean Somatic Embryos in Soybean Somatic Embryos

The present example describes the expression of pKR1337 (SEQ ID NO:273,Example 29), comprising Lipomyces starkeyi diacylglycerideacyltransferase 2 (LS_DGAT2), pKR1256 (SEQ ID NO:98; Example 16),comprising Yarrowia DGAT2 (YL_DGAT2), pKR1334 (SEQ ID NO:280, Example30), comprising Mortierella diacylglyceride acyltransferase 1 (Ma_DGAT1)and pKR1323 (SEQ ID NO:289, Example 30), comprising Yarrowia DGAT1 codonoptimized for expression in soy (YL_DGAT1cod2), in soy somatic embryos(Experiment numbers MSE2333, MSE2334, MSE2332 and MSE2331,respectively).

Events from each experiment were selected, somatic embryos were maturedin SHaM and oil concentrations and fatty acid profiles were determinedas described in Example 5. Results for MSE2331, MSE2332, MSE2333 and2334 are shown in Table 61, Table 62, Table 63 and Table 64,respectively.

TABLE 61 Oil concentrations and fatty acid profiles for events fromMSE2331 MSE2331 (YL DGAT1cod2) Event 16:0 18:0 18:1 18:2 18:3 % Oil2331-18 11.2 5.8 36.5 41.0 5.5 19.5 2331-29 11.3 5.6 41.7 36.8 4.6 18.02331-27 11.1 5.5 37.1 40.6 5.7 17.5 2331-14 11.6 4.6 32.9 43.7 7.2 16.32331-5 13.3 4.1 28.4 46.6 7.6 16.1 2331-13 12.5 5.7 34.9 39.9 7.0 15.62331-21 12.8 3.8 27.4 47.6 8.4 15.4 2331-16 11.7 4.9 34.9 41.2 7.2 14.42331-2 11.6 5.2 32.4 43.0 7.7 14.4 2331-17 11.0 5.4 35.4 41.0 7.3 14.32331-11 12.4 4.2 28.6 45.1 9.7 13.8 2331-24 11.8 7.4 37.0 36.9 6.9 12.42331-19 13.0 6.1 32.9 39.7 8.2 12.2 2331-9 13.6 5.3 29.3 42.8 9.0 12.22331-20 14.8 4.6 20.9 48.5 11.3 11.8 2331-28 14.2 5.2 25.5 45.7 9.3 11.72331-8 14.6 4.0 24.3 46.2 10.9 11.6 2331-15 15.6 3.8 20.3 48.3 12.0 11.52331-7 14.4 3.9 20.6 50.3 10.9 11.4 2331-4 14.0 4.2 25.1 47.4 9.4 11.22331-31 12.3 6.2 31.3 40.9 9.3 10.4 2331-26 13.4 4.0 16.4 54.3 11.8 10.32331-30 13.3 8.0 34.7 36.0 8.1 10.1 2331-12 13.8 6.4 29.1 40.5 10.1 9.72331-6 13.4 6.9 32.7 38.0 9.0 9.5 2331-1 14.3 5.6 31.3 36.9 11.8 8.22331-23 15.8 4.4 19.2 46.6 14.0 8.1 2331-10 16.1 4.3 20.9 44.2 14.5 7.82331-22 15.2 6.7 22.0 43.1 13.1 7.6 2331-25 16.9 5.7 22.1 40.1 15.2 5.52331-3 15.3 5.5 24.6 39.0 15.6 4.6 Avg. 13.4 5.3 28.7 43.0 9.6 12.0

TABLE 62 Oil concentrations and fatty acid profiles for events fromMSE2332 MSE2332 (MA DGAT1) Event 16:0 18:0 18:1 18:2 18:3 % Oil 2332-3116.0 6.7 22.9 38.9 15.6 18.9 2332-15 11.6 5.8 38.4 37.5 6.6 15.1 2332-2916.5 5.1 21.3 41.2 15.9 13.9 2332-9 12.4 4.5 33.2 41.4 8.6 13.8 2332-1412.1 5.5 36.9 38.4 7.2 13.6 2332-22 12.3 6.5 36.0 37.3 7.9 13.2 2332-2612.0 5.3 36.8 38.1 7.7 13.2 2332-20 16.9 5.2 17.7 43.1 17.1 12.7 2332-2512.7 5.7 35.8 38.3 7.6 12.5 2332-21 13.0 6.8 34.4 37.8 8.0 12.0 2332-613.2 5.6 34.1 38.0 9.1 11.5 2332-24 13.0 6.5 35.5 37.5 7.5 11.3 2332-2313.7 6.4 33.7 37.3 8.9 11.1 2332-2 13.0 5.5 33.3 38.5 9.7 10.5 2332-113.5 5.6 31.5 39.4 10.0 10.0 2332-19 14.0 6.2 31.8 38.1 9.9 9.8 2332-1114.4 5.6 28.8 40.6 10.7 9.4 2332-7 15.1 5.0 26.6 42.6 10.7 9.1 2332-1315.6 4.9 23.2 43.5 12.8 8.8 2332-18 15.3 4.5 21.4 45.4 13.5 8.1 2332-816.1 4.3 19.7 46.1 13.8 7.9 2332-16 16.7 4.2 20.8 43.5 14.8 7.1 2332-1215.6 6.5 23.7 39.8 14.4 6.8 2332-4 16.8 5.2 20.5 41.3 16.2 6.1 2332-1716.4 5.5 23.0 40.1 15.0 6.0 2332-28 17.1 5.4 23.0 37.1 17.4 5.5 2332-1016.9 4.6 18.3 41.7 18.5 5.3 2332-3 16.2 5.5 20.6 41.9 15.8 5.3 2332-517.1 5.1 19.6 39.7 18.5 4.6 2332-30 11.9 3.7 34.4 41.6 8.3 4.5 2332-2715.9 5.8 19.7 39.9 18.7 4.1 Avg. 14.6 5.4 27.6 40.2 12.1 9.7

TABLE 63 Oil concentrations and fatty acid profiles for events fromMSE2333 MSE2333 (LS DGAT2) Event 16:0 18:0 18:1 18:2 18:3 % Oil 2333-215.5 6.8 28.2 38.0 11.5 12.2 2333-23 13.8 5.2 28.7 43.1 9.2 11.6 2333-2713.4 6.0 35.3 37.2 8.1 10.5 2333-8 16.2 5.1 21.1 40.8 16.7 10.3 2333-2114.4 6.1 30.9 39.5 9.1 9.3 2333-20 14.1 7.0 32.3 37.0 9.7 9.3 2333-110.3 4.3 44.0 36.3 5.1 8.6 2333-29 15.5 6.0 28.0 39.2 11.3 8.4 2333-1414.2 5.3 27.8 40.7 12.0 8.2 2333-17 14.6 6.5 32.4 36.1 10.4 7.6 2333-2815.0 6.8 30.1 37.0 11.2 7.6 2333-26 16.8 4.8 21.8 43.3 13.4 7.3 2333-1815.3 5.4 29.7 37.5 12.1 6.8 2333-16 15.4 5.5 25.2 39.9 13.9 6.7 2333-1116.4 7.2 26.0 37.3 13.1 6.5 2333-24 13.9 4.9 24.4 41.9 14.8 6.3 2333-1515.4 5.4 28.2 37.7 13.3 6.2 2333-4 12.4 5.7 33.8 39.8 8.3 5.6 2333-517.0 5.5 19.0 41.4 17.2 5.4 2333-10 16.0 6.0 21.7 40.9 15.4 5.4 2333-1215.6 5.5 25.7 36.7 16.5 5.4 2333-31 15.4 5.7 26.7 37.9 14.2 5.1 2333-716.4 5.4 20.8 41.2 16.2 5.1 2333-19 16.2 6.7 26.6 36.6 14.0 4.9 2333-3015.9 5.5 25.9 37.6 15.1 4.8 2333-6 16.9 5.6 22.6 38.8 16.1 4.6 2333-2515.4 5.6 21.7 39.4 17.9 4.4 2333-3 13.4 5.1 31.4 40.9 9.1 4.4 2333-913.9 6.1 31.2 41.2 7.7 3.9 2333-13 17.2 4.5 15.8 41.5 21.0 3.8 2333-2216.6 5.6 21.4 37.3 19.1 2.9 Avg. 15.1 5.7 27.0 39.2 13.0 6.7

TABLE 64 Oil concentrations and fatty acid profiles for events fromMSE2334 MSE2334 (YL DGAT2) Event 16:0 18:0 18:1 18:2 18:3 % Oil 2334-2511.8 6.0 36.3 38.4 7.4 12.9 2334-12 12.5 6.9 37.9 36.2 6.6 12.5 2334-513.7 4.9 31.7 41.7 7.9 12.1 2334-3 12.4 5.3 38.6 36.4 7.3 11.1 2334-1912.5 6.0 33.0 38.2 10.3 10.8 2334-4 13.0 5.1 33.7 39.2 9.0 10.7 2334-2413.1 5.8 33.4 37.3 10.4 10.1 2334-1 15.0 5.0 29.4 40.5 10.2 9.5 2334-1813.8 5.1 30.1 40.3 10.6 9.3 2334-15 14.6 6.0 31.3 37.6 10.5 8.8 2334-1713.7 5.4 32.5 37.0 11.4 8.4 2334-30 15.3 5.6 29.4 37.4 12.2 7.9 2334-1615.2 6.5 29.6 37.0 11.8 7.3 2334-21 15.4 7.5 28.8 36.5 11.8 7.2 2334-1114.5 6.8 33.7 34.0 11.0 6.7 2334-28 14.9 4.5 27.2 39.1 14.3 6.5 2334-2715.8 5.8 28.4 37.1 12.9 6.3 2334-14 16.1 7.7 27.6 35.4 13.1 6.3 2334-2615.9 6.4 24.1 40.2 13.4 6.2 2334-9 16.6 4.1 18.3 42.3 18.8 6.1 2334-2017.3 4.5 19.2 41.6 17.4 5.6 2334-8 15.2 5.0 27.8 36.4 15.6 5.6 2334-2216.3 6.1 26.4 36.5 14.6 5.5 2334-2 15.8 4.6 22.3 40.4 16.9 4.9 2334-1017.0 5.0 22.3 38.8 16.9 4.9 2334-29 18.7 3.6 9.5 47.9 20.3 4.8 2334-716.5 5.6 23.0 36.9 17.9 4.7 2334-23 16.6 4.8 17.3 41.5 19.9 4.6 2334-619.3 6.1 23.5 36.4 14.6 4.0 2334-13 18.5 5.6 20.5 37.1 18.4 3.3 Avg.15.2 5.6 27.6 38.5 13.1 7.5

LS_DGAT2 increases oil and oleic acid concentration in somatic embryosto amounts similar to YL DGAT2. YL DGAT1(cod2) and MA DGAT1 increaseoleic similarly to YL DGAT2 and have higer oil increases with YLDGAT1(cod2) having the highest oil.

Example 38 Functional Analysis of DGAT2s from Yarrowia lipolyitica andPhaffia rhodozyma and DGAT1s from Yarrowia lipolytica and Lipomycesstarkeyi in Soybean Somatic Embryos

The present example describes the expression of pKR1372 (SEQ ID NO:294,Example 31), comprising Phaffia rhodozyma diacylglycerideacyltransferase 2 (PR_DGAT2), pKR1256 (SEQ ID NO:98; Example 16),comprising Yarrowia DGAT2 (YL_DGAT2), pKR1375 (SEQ ID NO:300, Example32), comprising Lipomyces diacylglyceride acyltransferase 1 (LS_DGAT1)and pKR1323 (SEQ ID NO:289, Example 30), comprising Yarrowia DGAT1 codonoptimized for expression in soy (YL_DGAT1cod2), in soy somatic embryos(Experiment numbers MSE2351, MSE2349, MSE2352 and MSE2350,respectively).

Events from each experiment were selected, somatic embryos were maturedin SHaM and oil concentrations and fatty acid profiles were determinedas described in Example 5. Results for MSE2349, MSE2350, MSE2351 and2352 are shown in Table 65, Table 66, Table 67 and Table 68,respectively.

TABLE 65 Oil concentrations and fatty acid profiles for events fromMSE2349 MSE2349 (YL DGAT2) Event 16:0 18:0 18:1 18:2 18:3 % Oil 2349-310.3 4.1 42.5 37.0 6.1 17.3 2349-29 11.8 4.4 32.6 43.2 8.2 15.1 2349-1711.5 4.0 34.5 42.1 8.0 15.0 2349-22 10.8 4.9 34.1 42.2 8.0 14.6 2349-1111.5 4.9 34.1 41.9 7.6 14.1 2349-27 13.0 4.5 31.0 43.0 8.5 13.6 2349-2613.6 3.7 28.0 45.6 9.1 12.7 2349-14 12.9 5.0 30.0 43.1 9.1 12.0 2349-2514.7 4.4 20.6 49.9 10.4 11.9 2349-2 12.4 6.4 39.5 33.9 7.8 11.1 2349-515.3 3.6 20.7 48.2 12.3 10.8 2349-24 14.5 5.2 26.0 43.5 10.8 10.52349-21 12.6 6.9 35.2 36.0 9.4 10.3 2349-6 13.0 6.1 33.6 38.2 9.2 10.32349-16 14.6 4.5 23.8 46.1 11.1 10.2 2349-1 12.8 5.1 32.0 39.7 10.4 9.92349-23 15.1 4.0 23.0 46.6 11.3 9.9 2349-20 14.1 5.1 23.2 46.0 11.6 9.72349-18 14.2 4.4 24.4 45.0 12.1 9.2 2349-19 14.5 6.1 26.8 41.7 10.9 9.22349-28 13.2 7.4 33.4 35.7 10.3 9.2 2349-4 14.7 4.4 25.0 44.7 11.2 8.62349-12 17.9 3.9 17.0 47.7 13.5 8.6 2349-10 15.7 6.7 22.8 41.6 13.1 8.32349-15 17.6 5.1 21.9 43.5 11.9 8.1 2349-8 13.8 5.4 22.5 43.0 15.2 8.12349-30 14.8 4.5 27.7 39.6 13.6 8.0 2349-9 14.9 6.5 26.9 39.7 12.1 7.72349-13 15.0 5.1 25.0 39.6 15.3 6.7 2349-7 16.3 4.5 20.8 42.1 16.3 5.7Avg. 13.9 5.0 27.9 42.3 10.8 10.5

TABLE 66 Oil concentrations and fatty acid profiles for events fromMSE2350 MSE2350 (YL DGAT1cod2) Event 16:0 18:0 18:1 18:2 18:3 % Oil2350-4 10.7 3.9 34.7 44.7 5.9 18.6 2350-20 9.9 3.9 45.0 34.9 6.3 17.72350-6 10.5 4.7 40.2 38.8 5.7 17.3 2350-29 11.0 4.7 37.8 40.5 6.0 16.62350-5 10.8 5.3 37.0 39.8 7.1 13.8 2350-1 11.4 5.4 37.1 38.5 7.5 13.72350-23 12.9 5.4 32.9 41.3 7.6 12.4 2350-17 12.3 7.5 35.9 37.0 7.4 12.42350-24 14.2 4.1 22.7 47.9 11.0 11.7 2350-3 14.0 5.4 30.6 40.3 9.6 11.72350-12 13.4 6.6 27.8 42.9 9.4 11.3 2350-18 14.4 4.1 21.1 49.2 11.1 11.12350-14 14.6 3.2 21.3 49.3 11.7 10.9 2350-30 13.8 6.9 29.1 40.3 9.9 10.62350-26 14.2 5.8 26.3 43.3 10.5 10.5 2350-21 12.9 5.8 28.5 42.7 10.210.1 2350-16 15.2 5.2 22.3 46.3 11.0 9.5 2350-19 13.7 5.2 27.7 41.7 11.69.5 2350-13 14.7 5.1 24.6 43.9 11.8 9.0 2350-7 14.7 6.4 25.8 41.2 11.98.9 2350-2 13.3 5.6 31.9 38.0 11.2 8.2 2350-27 15.9 5.3 23.0 42.7 13.17.9 2350-28 16.4 5.9 27.0 38.3 12.4 7.6 2350-22 15.3 6.4 23.6 42.1 12.57.6 2350-25 13.8 4.8 24.0 42.7 14.6 7.5 2350-31 16.6 4.5 21.1 43.8 14.07.3 2350-11 14.8 5.9 22.3 43.0 14.0 7.2 2350-8 16.1 5.4 22.6 40.5 15.36.5 2350-10 15.5 5.9 22.1 41.3 15.2 6.0 2350-9 15.4 5.8 20.6 41.6 16.65.2 2350-15 15.1 4.5 17.1 43.1 20.1 5.1 Avg. 13.8 5.3 27.9 42.0 11.010.4

TABLE 67 Oil concentrations and fatty acid profiles for events fromMSE2351 MSE2351 (PR DGAT2) Event 16:0 18:0 18:1 18:2 18:3 % Oil 2351-2815.2 4.2 21.9 48.7 10.0 12.3 2351-9 14.8 3.9 22.0 48.0 11.3 12.0 2351-2414.8 4.3 21.9 49.1 9.9 11.2 2351-11 15.3 5.0 23.8 45.6 10.3 10.8 2351-2514.4 6.0 23.9 44.6 11.2 10.6 2351-21 15.3 5.6 24.4 44.0 10.7 10.52351-30 14.7 5.9 25.3 43.9 10.2 10.1 2351-1 13.9 6.8 25.1 44.2 10.0 10.12351-10 15.3 7.1 24.8 42.8 10.0 10.1 2351-13 14.9 7.0 25.9 42.8 9.4 10.02351-23 14.7 6.6 25.3 42.6 10.7 9.6 2351-18 15.3 5.0 23.0 46.4 10.3 9.52351-12 15.1 4.1 17.4 50.7 12.8 9.2 2351-3 15.4 7.2 25.1 42.1 10.3 8.82351-15 15.1 4.9 22.6 46.1 11.3 8.7 2351-5 15.7 7.4 23.5 42.2 11.1 8.62351-16 15.6 5.7 23.4 43.9 11.4 8.4 2351-6 14.8 6.1 25.7 42.5 10.9 8.02351-20 15.6 6.4 25.3 41.2 11.6 8.0 2351-29 17.6 5.4 19.9 43.9 13.2 8.02351-22 15.1 7.0 25.8 40.4 11.7 7.9 2351-4 15.5 6.6 23.1 41.6 13.1 7.82351-2 15.4 6.6 24.3 41.4 12.4 7.7 2351-8 15.6 6.6 24.9 39.1 13.8 6.72351-26 15.8 6.4 23.0 40.3 14.5 6.7 2351-14 11.8 4.0 20.7 50.0 13.4 6.32351-17 17.3 5.6 21.3 39.8 16.0 5.3 2351-19 16.4 6.4 23.0 40.1 14.2 5.32351-7 14.5 5.9 21.6 40.3 17.6 4.9 2351-27 16.5 6.4 22.8 40.1 14.1 4.9Avg. 15.2 5.9 23.4 43.6 11.9 8.6

TABLE 68 Oil concentrations and fatty acid profiles for events fromMSE2352 MSE2352 (LS DGAT1) Event 16:0 18:0 18:1 18:2 18:3 % Oil 2352-413.2 4.4 28.1 45.4 8.9 14.0 2352-6 12.4 5.8 30.3 43.3 8.1 13.6 2352-2714.3 7.6 32.9 37.4 7.9 12.9 2352-8 14.9 4.5 21.3 49.0 10.4 12.1 2352-2413.2 7.5 31.0 39.6 8.7 12.1 2352-5 13.7 7.8 29.9 40.0 8.6 12.0 2352-3014.4 5.9 30.0 40.6 9.1 11.9 2352-29 14.0 6.1 31.5 39.3 9.1 11.7 2352-2214.4 4.7 24.6 46.4 9.9 11.6 2352-28 14.4 7.4 28.3 40.8 9.1 11.5 2352-1214.4 7.5 29.0 39.4 9.7 11.1 2352-19 14.5 6.1 30.5 38.7 10.2 10.9 2352-714.2 7.0 24.9 44.2 9.7 10.9 2352-21 15.5 6.8 25.4 42.0 10.3 10.6 2352-114.7 8.0 27.7 39.5 10.1 10.2 2352-9 15.0 5.7 22.7 44.5 12.2 9.3 2352-1312.6 5.4 27.8 42.8 11.3 9.2 2352-11 14.7 7.8 26.8 40.2 10.6 9.1 2352-1616.3 6.0 23.5 42.0 12.2 9.1 2352-18 15.2 5.9 26.7 40.4 11.9 8.7 2352-1415.8 5.2 21.4 44.8 12.8 8.5 2352-15 15.2 6.2 23.5 42.2 12.8 8.3 2352-1016.2 6.3 24.5 39.8 13.2 7.9 2352-25 15.4 5.3 26.5 40.4 12.4 7.8 2352-2618.9 5.0 18.5 45.1 12.6 7.7 2352-23 15.2 8.1 25.5 39.4 11.8 7.4 2352-316.1 7.0 23.8 39.8 13.3 7.0 2352-17 15.0 6.3 24.7 39.9 14.1 6.5 2352-3117.1 6.3 22.0 41.5 13.1 6.3 2352-32 16.8 7.2 26.8 37.2 12.1 6.1 2352-216.5 7.2 23.1 39.6 13.7 5.9 2352-20 16.0 7.2 26.5 36.3 14.0 5.2 Avg.15.0 6.4 26.2 41.3 11.1 9.6

LS_DGAT1 and YL_DGAT1(cod2) increase oil and oleic acid concentration insomatic embryos to amounts similar to YL DGAT2. PR_DGAT2 increases oiland oleic to a slightly lesser extent than YL_DGAT2. It is noted thatabsolute concentrations (expressed as averages) may vary using the samegenes but in different separate experiments conducted at differenttimes. For example, YL_DGAT2 gave an average oil concentration of 7.5%in MSE2334 (Example 37) but gives 10.5% in MSE2349. In all cases,average % Oil for any individual gene in an experiment can be comparedto that obtained for YL_DGAT2 which is run in all experiments as acontrol and thus normalizes results between expermimental sets.

Example 39 Expression and Functional Analysis of DGAT2s from Yarrowialipolyitica, Cryptococcus curvatus, Mucor circinelloides and Candidazeylanoides in Soybean Somatic Embryos

The present example describes construction of pKR1392, comprisingCandida zeylanoides diacylglyceride acyltransferase 2 (CZ_DGAT2);pKR1409, comprising Mucor circinelloides diacylglyceride acyltransferase2 (MC_DGAT2); and pKR1427, comprising Cryptococcus curvatusdiacylglyceride acyltransferase 2 (CC_DGAT2) and expression in somaticembryos. Control vector pKR1256 (SEQ ID NO:98; Example 16), comprisingYarrowia DGAT2 (YL_DGAT2), was also expressed for comparison.

Construction of pKR1392, comprising Cz DGAT2

CZ_DGAT2 (SEQ ID NO:175, Example 21) was amplified from an isolatedcosmid containing the genomic DNA fragment (described in Example 21)with oligonucleotide primers CzDGAT2-5 (SEQ ID NO:352) and CzDGAT2-3(SEQ ID NO:353), using the Phusion™ High-Fidelity DNA Polymerase (Cat.No. F553S, Finnzymes Oy, Finland) following the manufacturer's protocol.The resulting DNA fragment was cloned into the pCR-Blunt® cloning vectorusing the Zero Blunt® PCR Cloning Kit (Invitrogen Corporation),following the manufacturer's protocol, to produce pHD39 (SEQ ID NO:354).

The NotI fragment of pKRHD39 (SEQ ID NO:354), containing CZ_DGAT2, wascloned into the NotI site of pKR72 (SEQ ID NO:26; Example 4) to producepKR1392 (SEQ ID NO:355).

Construction of pKR1409, comprising MC_DGAT2

MC_DGAT2 (SEQ ID NO:233, Example 26) was amplified from cDNA describedin Example 26 with oligonucleotide primers oMcDG2-1 (SEQ ID NO:356) andoMcDG2-2 (SEQ ID NO:357), using the Phusion™ High-Fidelity DNAPolymerase (Cat. No. F553S, Finnzymes Oy, Finland) following themanufacturer's protocol. The resulting DNA fragment was cloned into thepCR-Blunt® cloning vector using the Zero Blunt® PCR Cloning Kit(Invitrogen Corporation), following the manufacturer's protocol, toproduce pKR1408 (SEQ ID NO:358). The nucleotide sequence of MC_DGAT2from pKR1408 is set forth in SEQ ID NO:359 and has one nt changecompared to SEQ ID NO:233 which does not alter the aa sequence.

The NotI fragment of pKR1408 (SEQ ID NO:358), containing MC_DGAT2, wascloned into the NotI site of pKR72 (SEQ ID NO:26; Example 4) to producepKR1409 (SEQ ID NO:360).

Construction of pKR1427, comprising CC_DGAT2

The plasmid containing the CC_DGAT2 gene described in Example 36 wasdigested with NotI and the fragment containing CC_DGAT2 was cloned intothe NotI site of pKR72 (SEQ ID NO:26; Example 4) to produce pKR1427 (SEQID NO:361).

Expression of CZ_DGAT2, MC DGAT2 and CC DGAT2 in soy somatic embryos

Soybean embryogenic suspension culture (cv. Jack) was transformed withpKR1392 (SEQ ID NO:355), having experiment number MSE2451; with pKR1409(SEQ ID NO:360), having experiment number MSE2452 and with pKR1427 (SEQID NO:361), having experiment number MSE2453. Control vector pKR1256(SEQ ID NO:98; Example 16), comprising Yarrowia DGAT2 (YL_DGAT2), wasalso transformed alone in a similar way for a control and has experimentnumber MSE2454. Events were selected and somatic embryos matured in SHaMas described in Example 5. Oil concentrations and fatty acid profileswere determined as described in Example 5 for MSE2451, MSE2452, MSE 2453and MSE2454 and results for each experiment are shown in Table 69, Table70, Table 71 and Table 72, respectively.

TABLE 69 Oil concentrations and fatty acid profiles for events fromMSE2451 MSE2451 (CZ DGAT2) Event 16:0 18:0 18:1 18:2 18:3 % Oil 2451-1316.2 5.3 17.0 38.1 23.4 7.0 2451-7 16.8 4.8 13.5 41.2 23.6 5.8 2451-2615.3 6.3 21.4 40.3 16.7 5.3 2451-25 15.7 5.5 16.6 39.4 22.8 5.2 2451-2415.4 5.1 15.0 40.9 23.6 5.0 2451-31 15.6 5.7 18.7 42.4 17.5 4.9 2451-1415.7 6.3 22.7 37.9 17.4 4.9 2451-12 16.6 6.2 21.1 38.5 17.6 4.7 2451-3016.7 5.2 17.0 41.4 19.7 4.6 2451-3 15.9 6.3 21.1 39.3 17.4 4.5 2451-914.4 6.6 24.8 36.4 17.9 4.4 2451-17 16.7 5.8 18.6 41.2 17.8 4.4 2451-1514.4 7.7 24.6 37.7 15.6 4.3 2451-10 16.2 6.2 20.6 38.8 18.2 4.3 2451-215.2 5.3 16.5 42.1 20.9 4.1 2451-21 16.9 5.8 18.8 36.6 21.9 4.0 2451-416.8 6.7 22.6 36.1 17.8 4.0 2451-16 17.1 6.2 16.4 38.3 22.0 3.9 2451-2816.2 5.3 17.4 40.6 20.5 3.9 2451-1 17.1 5.1 15.5 40.0 22.3 3.8 2451-617.8 4.8 11.3 41.3 24.9 3.8 2451-22 15.2 7.4 15.6 40.0 21.6 3.7 2451-2016.0 6.2 21.4 37.7 18.7 3.7 2451-8 16.3 5.2 17.0 39.8 21.8 3.7 2451-1816.2 5.5 19.9 40.0 18.3 3.5 2451-23 16.8 5.2 16.5 39.6 21.9 3.3 2451-2916.7 5.0 16.5 39.2 22.7 3.3 2451-5 17.2 6.1 18.0 39.0 19.7 3.2 2451-2716.4 5.2 16.2 39.1 23.2 3.1 2451-11 16.2 5.0 15.4 41.0 22.5 3.0 2451-1916.7 5.2 15.8 38.7 23.6 2.7 Avg. 16.2 5.7 18.2 39.4 20.4 4.2

TABLE 70 Oil concentrations and fatty acid profiles for events fromMSE2452 MSE2452 (MC DGAT2) Event 16:0 18:0 18:1 18:2 18:3 % Oil 2452-616.6 5.3 20.9 41.0 16.2 5.6 2452-22 15.6 6.6 24.7 39.2 13.9 5.5 2452-1316.8 7.1 28.1 35.1 13.0 5.4 2452-12 16.8 5.5 22.9 39.1 15.8 5.1 2452-316.8 5.0 22.0 39.9 16.3 5.1 2452-25 16.1 4.7 23.7 38.4 17.1 5.0 2452-2916.2 5.1 25.2 36.9 16.7 4.8 2452-20 17.1 5.3 20.7 38.1 18.8 4.8 2452-1116.8 5.2 24.6 37.2 16.3 4.8 2452-2 17.0 5.3 22.4 38.0 17.2 4.7 2452-816.5 4.4 22.4 39.3 17.4 4.7 2452-4 17.5 4.9 21.5 37.3 18.8 4.7 2452-2817.3 4.4 19.2 40.5 18.7 4.6 2452-1 16.3 5.6 26.5 35.1 16.5 4.5 2452-3117.6 5.7 22.6 38.0 16.2 4.4 2452-27 16.5 5.7 25.5 35.7 16.6 4.4 2452-2318.5 6.3 23.1 36.0 16.0 4.3 2452-21 16.9 5.8 21.7 38.5 17.1 4.2 2452-516.4 5.3 24.1 36.1 18.1 4.0 2452-16 17.8 5.7 21.9 37.8 16.8 3.9 2452-1417.3 5.3 24.3 35.8 17.3 3.9 2452-26 16.8 4.4 18.0 41.4 19.3 3.9 2452-1816.7 4.5 16.4 39.0 23.4 3.8 2452-24 19.4 4.8 13.7 40.1 22.0 3.7 2452-916.8 5.1 13.6 42.8 21.7 3.6 2452-7 18.7 4.8 16.7 37.8 21.8 3.4 2452-1517.5 5.3 18.4 38.2 20.7 3.0 2452-30 19.3 5.5 17.8 37.9 19.5 3.0 2452-1017.1 5.6 19.4 37.1 20.8 2.5 Avg. 17.1 5.3 21.4 38.2 17.9 4.3

TABLE 71 Oil concentrations and fatty acid profiles for events fromMSE2453 MSE2453 (CC DGAT2) Event 16:0 18:0 18:1 18:2 18:3 % Oil 2453-1116.1 6.0 22.5 40.2 15.3 5.6 2453-25 17.2 5.0 17.3 42.8 17.6 5.1 2453-1216.3 6.1 23.0 39.1 15.4 5.1 2453-29 16.9 4.7 16.2 41.1 21.1 5.1 2453-1517.5 4.2 12.2 45.2 20.9 4.7 2453-13 18.8 5.6 20.5 37.8 17.3 4.5 2453-217.3 5.4 19.7 38.0 19.6 4.4 2453-26 16.5 4.8 18.6 38.0 22.1 4.3 2453-717.6 5.4 19.4 39.7 18.0 4.3 2453-20 17.3 4.7 16.0 39.9 22.1 4.1 2453-818.2 6.1 22.9 35.9 16.8 4.1 2453-6 17.1 5.0 20.2 38.2 19.5 4.1 2453-2417.7 5.0 14.9 40.9 21.5 4.1 2453-14 17.6 5.1 19.8 38.6 18.9 4.0 2453-2219.2 4.4 12.6 39.4 24.5 4.0 2453-28 18.2 5.4 21.5 37.0 17.9 4.0 2453-1818.0 5.7 16.5 39.3 20.4 3.9 2453-21 19.6 5.9 19.1 37.0 18.4 3.9 2453-1017.4 5.1 15.4 39.8 22.4 3.8 2453-17 18.0 5.6 15.9 40.8 19.7 3.7 2453-517.2 4.0 11.1 43.4 24.1 3.5 2453-23 18.1 4.9 15.1 41.4 20.6 3.4 2453-317.7 5.0 15.5 39.7 22.0 3.3 2453-9 18.6 5.6 18.3 36.0 21.6 3.2 2453-417.5 5.0 15.3 40.1 22.0 3.2 2453-16 17.2 5.7 25.1 32.1 20.0 2.8 2453-3016.9 4.6 17.7 37.5 23.2 2.6 2453-19 20.6 4.4 12.0 38.4 24.5 2.3 Avg.17.7 5.2 17.6 39.2 20.3 4.0

TABLE 72 Oil concentrations and fatty acid profiles for events fromMSE2454 MSE2454 (YL DGAT2) Event 16:0 18:0 18:1 18:2 18:3 % Oil 2454-2712.2 6.0 35.5 36.8 9.4 9.5 2455-5 13.9 5.9 32.6 36.9 10.8 8.2 2454-1313.6 5.8 29.4 38.9 12.3 8.0 2454-29 14.1 5.4 29.7 37.6 13.2 7.9 2454-2614.5 6.3 33.8 33.9 11.5 7.3 2454-24 14.9 5.9 28.2 38.6 12.4 7.0 2454-1715.3 5.1 27.0 38.7 13.9 6.6 2454-9 14.1 6.0 31.0 36.8 12.1 6.6 2454-1514.6 5.9 23.0 39.5 16.9 6.4 2454-10 16.1 6.4 27.4 36.2 14.0 5.8 2454-217.0 5.2 22.0 40.3 15.5 5.8 2454-1 15.8 6.0 26.4 37.4 14.4 5.7 2455-417.2 5.7 23.6 37.8 15.8 5.5 2454-7 15.4 5.6 23.6 39.1 16.4 5.4 2454-815.8 5.5 28.8 35.8 14.0 5.3 2454-18 17.4 6.3 21.7 37.6 17.0 5.2 2454-317.8 5.9 23.0 38.3 14.9 5.1 2454-16 16.5 5.4 21.0 40.8 16.3 5.0 2454-2317.3 4.5 19.5 40.4 18.3 4.9 2454-11 16.8 5.6 24.3 36.3 17.0 4.8 2454-2516.8 6.0 20.9 38.2 18.1 4.8 2455-7 17.3 5.4 24.2 38.1 15.0 4.7 2454-417.5 6.7 21.9 37.7 16.2 4.7 2455-6 17.2 5.3 19.9 39.3 18.3 4.7 2454-1416.5 5.8 23.1 37.4 17.2 4.4 2454-6 17.6 6.1 25.6 35.3 15.4 4.4 2454-2116.6 4.4 20.9 38.1 20.0 3.9 2454-19 18.4 5.0 17.6 40.4 18.6 3.7 2454-3016.6 4.1 15.0 40.9 23.3 3.6 2454-31 17.3 4.1 11.4 43.3 23.8 3.6 2454-2817.5 5.0 19.7 37.5 20.2 3.5 2454-22 17.5 4.8 19.6 39.7 18.4 3.4 Avg.16.2 5.5 24.1 38.2 16.0 5.5

The wild-type CZ_DGAT2, MC_DAGT2 and CC_DGAT2 do not appear to affectoil or oleic acid concentration in somatic embryos when compared to YLDGAT2.

Example 40 Expression Optimized DGAT2 Gene Sequences Encoding a CC_DGAT2Protein Variant

The nucleotide sequence encoding the DGAT2 protein from Cryptococcuscurvatus described herein (see previous Example 36) was re-designed foroptimized expression in soybean seed using methods similar to thosedescribed in Wu, G et al. Nucleic Acids Research (2007), 35: D76-D79;Villalobos, A. et al. BMC Bioinformatics (2006), 7 No pp. given; Wu, G.et al. Protein Expression and Purification (2006), 47: 441-445;Richardson, S. M. et al. Genome Research (2006), 16: 550-556; Jayaraj,S. et al. Nucleic Acids Research (2005) 33: 3011-3016. DNA moleculeswere synthesized by either Codon Devices (MA, USA) or by GENEART AG(Regensburg, Germany). The expression-optimized DNA sequence and DGAT2protein encoded by said DNA sequence are shown in SEQ ID NO:362 and SEQID NO:363, respectively. The protein encoded by the codon-optimizedCC_DGAT2 sequence contains the motif FxxPxFR that was altered fromFxxPxYR present in the native DGAT2 protein.

Example 41 Cloning Codon-Optimized DGAT Sequences for Expression in Soy

The present example describes the cloning of DGAT sequences which werecodon-optimized, and motif-altered in the case of DGAT2s, for expressionin soy into soy expression vectors.

All DGAT sequences that were codon-optimized for expression in soy, asdescribed in Examples 33, 34 and 40, were synthesized with NotIrestriction enzyme sites flanking the codon-optimized gene sequence(before start codon and after stop codon). In addition, threenucleotides (ACC) were added between with NotI at the 5′ end ofcodon-optimized gene and the ATG start codon in all cases.

The NotI fragments of each synthesized codon-optimized DGAT genesequence were cloned into the NotI site of pKR72 (SEQ ID NO:26; Example4) to produce soy expression vectors. The vector sequences for allwild-type and codon-optimized gene sequences are summarized in Table 73.

TABLE 73 Summary of wild-type and codon-optimized sequences and vectorsnt aa SEQ ID SEQ ID Expression Vector Gene NO: NO: Vector SEQ ID NO:TD_DGAT2A 133 135 pKR1324 121 TD_DGAT2Acod 303 304 pKR1422 364 TD_DGAT2B134 136 pKR1325 134 TD_DGAT2Bcod 305 306 N/A N/A PA_DGAT2 146 147pKR1332 260 PA_DGAT2cod 307 308 pKR1421 365 DH_DGAT2 161 162 pKR1328 161DH_DGAT2cod 309 310 pKR1420 366 CZ_DGAT2 175 176 pKR1392 355 CZ_DGAT2cod311 312 pKR1512 367 LS_DGAT2 271 272 pKR1337 273 LS_DGAT2cod 313 314pKR1415 368 MC_DGAT2 233 234 pKR1409 360 MC_DGAT2cod 315 316 pKR1513 369PR_DGAT2 293 215 pKR1372 294 PR_DGAT2cod 317 318 pKR1416 370 RG_DGAT2264 265 pKR1333 266 RG_DGAT2cod 319 320 pKR1423 371 MA_DGAT2 254 255pKR1335 256 MA_DGAT2cod 321 322 pKR1419 372 CC_DGAT2 350 351 pKR1427 361CC_DGAT2cod 362 363 pKR1522 373 LS_DGAT1 298 299 pKR1375 300 LS_DGAT1cod302 identical to pKR1514 374 wt MA_DGAT1 278 279 pKR1334 280 MA_DGAT1cod301 identical to pKR1511 375 wt

Example 42 Expression of Codon-Optimized and Wild-Type DGAT Sequences inSoy

The present example describes the expression and functionalcharacterization of wild-type and codon-optimized DGAT sequences is soysomatic embryos.

Soybean embryogenic suspension culture (cv. Jack) was transformed witheach of the soy expression vectors comprising wild-type orcodon-optimized DGATs described in Table 73 (Example 41).Transformations were carried out in sets comparing wild-type genes tocodon-optimized genes and generally four genes (2 wild-type, 2codon-optimized) were compared at once. Events were selected and somaticembryos matured in SHaM as described in Example 5. Oil concentrationsand fatty acid profiles were determined as described in Example 5.

Results for pKR1337 (LS_DGAT2; SEQ ID NO:273; Experiment MSE2411),pKR1415 (LS_DGAT2cod; SEQ ID NO:368; Experiment MSE2412), pKR1372(PR_DGAT2; SEQ ID NO:294; Experiment MSE2413) and pKR1416 (PR_DGAT2cod;SEQ ID NO:370; Experiment MSE2414) are shown in Tables 74, Table 75,Table 76 and Table 77, respectively.

TABLE 74 Oil concentrations and fatty acid profiles for events fromMSE2411 MSE2411 (LS DGAT2) Event 16:0 18:0 18:1 18:2 18:3 % Oil 2411-2415.7 5.0 27.3 41.9 10.2 10.8 2411-31 13.6 5.8 28.2 41.7 10.7 8.7 2411-3213.7 5.6 28.6 41.4 10.7 8.6 2411-12 14.0 6.5 29.8 39.0 10.7 8.5 2411-1716.6 5.6 21.3 42.9 13.6 7.7 2411-22 14.3 6.5 30.9 37.3 11.0 7.5 2411-2116.0 6.9 27.2 36.8 13.2 6.3 2411-30 15.7 6.4 23.1 41.1 13.8 6.2 2411-516.9 6.6 26.3 37.3 12.9 6.0 2411-29 14.7 5.3 27.5 37.7 14.9 6.0 2411-516.9 6.4 21.1 41.5 14.1 5.7 2411-13 16.4 6.6 24.1 39.7 13.3 5.3 2411-2717.0 5.1 21.1 40.1 16.6 5.2 2411-16 18.4 5.2 17.3 42.5 16.6 5.0 2411-2317.9 6.2 20.2 40.2 15.4 5.0 2411-6 16.6 4.8 18.2 42.7 17.7 5.0 2411-417.3 5.8 20.2 40.5 16.3 4.9 2411-20 15.4 5.9 25.3 38.0 15.4 4.9 2411-416.4 4.3 20.1 40.8 18.3 4.8 2411-2 17.3 5.0 18.4 41.4 17.9 4.7 2411-2617.0 5.9 19.1 40.6 17.4 4.6 2411-25 16.5 5.4 22.7 39.0 16.4 4.6 2411-1418.1 5.0 16.4 41.9 18.5 4.6 2411-15 16.7 5.8 23.2 37.9 16.4 4.5 2411-317.9 5.5 17.7 41.2 17.8 4.4 2411-1 16.3 5.0 19.9 40.4 18.4 4.4 2411-1017.6 5.4 20.7 40.2 16.2 4.4 2411-3 17.3 5.7 19.3 40.8 16.9 3.9 2411-2816.7 5.9 20.1 39.3 18.0 3.7 2411-18 17.3 4.5 16.1 41.4 20.7 3.6 2411-1118.0 5.0 16.4 44.3 16.3 2.9 2411-19 18.9 5.1 17.2 39.6 19.1 2.4 Avg.16.5 5.6 22.0 40.3 15.5 5.5

TABLE 75 Oil concentrations and fatty acid profiles for events fromMSE2412 MSE2412 (LS DGAT2cod) Event 16:0 18:0 18:1 18:2 18:3 % Oil2412-13 16.1 5.9 22.9 41.4 13.7 6.8 2412-29 16.2 6.6 23.0 41.1 13.1 6.62412-3 17.0 5.2 19.7 42.7 15.5 6.5 2412-9 16.2 6.2 22.9 41.2 13.5 6.22412-25 16.0 6.0 24.7 39.1 14.2 6.2 2412-26 16.6 5.8 21.8 42.0 13.8 6.12412-16 16.4 8.0 24.9 37.1 13.5 5.6 2412-23 16.1 6.5 23.3 40.0 14.0 5.52412-15 16.7 6.2 22.2 40.2 14.8 5.5 2412-5 16.2 6.7 27.3 36.9 12.9 5.42412-10 15.8 7.1 23.7 39.0 14.4 4.9 2412-4 17.3 3.2 12.9 44.9 21.8 4.82412-21 17.0 5.7 20.6 40.2 16.6 4.7 2412-30 16.6 5.9 22.6 39.8 15.0 4.72412-20 16.6 6.9 22.8 39.0 14.7 4.6 2412-28 16.6 5.1 17.1 42.7 18.5 4.62412-11 17.1 7.4 24.6 36.5 14.3 4.4 2412-31 17.7 4.4 14.9 43.5 19.5 4.42412-24 16.6 6.8 23.7 37.4 15.4 4.4 2412-8 17.9 5.6 17.2 44.0 15.3 4.42412-1 17.3 6.0 21.3 40.0 15.3 4.4 2412-7 17.8 6.1 20.8 40.2 15.1 4.32412-17 17.8 4.7 17.2 41.8 18.5 4.3 2412-18 17.4 4.4 15.1 42.9 20.3 4.32412-22 18.1 3.9 13.9 43.5 20.6 3.9 2412-19 17.5 4.9 14.8 42.5 20.3 3.92412-12 17.2 6.1 21.9 38.5 16.2 3.8 2412-6 17.2 6.0 22.8 38.0 15.9 3.72412-27 16.5 4.5 16.5 40.8 21.7 3.6 Avg. 16.9 5.8 20.6 40.6 16.2 4.9

TABLE 76 Oil concentrations and fatty acid profiles for events fromMSE2413 MSE2413 (PRDGAT2) Event 16:0 18:0 18:1 18:2 18:3 % Oil 2413-3014.7 5.2 20.3 47.2 12.5 9.4 2413-15 15.0 6.0 24.5 41.8 12.7 8.3 2413-2216.2 4.8 17.7 45.7 15.6 7.7 2413-7 16.2 5.1 18.8 44.5 15.3 7.5 2413-1615.7 5.6 22.0 42.5 14.1 7.5 2413-23 15.6 5.8 22.2 41.0 15.3 7.3 2413-1114.9 6.0 23.5 41.7 14.0 7.2 2413-31 15.6 8.7 17.9 42.9 14.8 6.1 2413-2816.6 6.3 23.8 38.2 15.0 6.1 2413-21 15.8 6.4 20.0 41.5 16.3 6.0 2413-2715.7 5.7 21.7 41.3 15.7 6.0 2413-4 16.4 5.0 18.2 43.5 17.0 5.8 2413-1916.0 5.4 22.5 40.3 15.8 5.7 2413-18 16.1 5.7 21.0 40.0 17.2 5.5 2413-1417.2 4.6 17.6 42.6 18.0 5.5 2413-13 16.7 5.5 19.7 41.5 16.6 5.4 2413-2416.6 5.9 19.6 41.3 16.5 5.3 2413-25 16.8 4.9 16.4 42.5 19.3 4.9 2413-216.6 5.3 19.8 40.8 17.6 4.7 2413-6 16.4 5.3 20.0 40.0 18.2 4.7 2413-2615.8 6.4 22.8 38.5 16.5 4.6 2413-20 16.0 6.4 22.2 38.5 16.8 4.6 2413-2916.1 6.1 20.9 38.0 18.9 4.4 2413-17 16.8 5.5 21.1 38.0 18.5 4.3 2413-1017.4 4.7 16.9 41.5 19.5 4.1 2413-8 17.2 4.8 16.2 40.9 20.9 3.9 2413-516.3 5.2 20.3 38.9 19.4 3.9 2413-3 16.1 5.5 21.8 38.0 18.6 3.8 2413-116.7 4.8 18.1 40.3 20.1 3.7 2413-12 16.8 5.0 18.7 39.8 19.8 3.5 2413-915.2 6.0 17.3 43.1 18.4 2.6 Avg. 16.2 5.6 20.1 41.2 16.9 5.5

TABLE 77 Oil concentrations and fatty acid profiles for events fromMSE2414 MSE2414 (PR DGAT2cod) Event 16:0 18:0 18:1 18:2 18:3 % Oil2414-12 15.8 4.5 18.9 46.3 14.6 8.7 2414-15 15.7 4.7 19.8 45.6 14.2 8.42414-6 14.5 4.5 25.4 40.5 15.1 8.3 2414-31 17.4 5.5 22.5 39.6 15.1 6.92414-32 15.2 4.8 26.5 39.2 14.4 6.7 2414-27 14.9 5.4 26.2 39.1 14.4 6.62414-8 15.1 4.9 27.2 38.8 14.0 6.5 2414-14 16.8 5.1 18.7 43.1 16.3 6.32414-26 15.5 4.6 24.1 40.2 15.5 6.2 2414-29 14.6 5.9 23.9 38.2 17.4 6.22414-9 15.6 4.7 26.9 38.0 14.8 6.1 2414-11 15.1 5.1 26.5 38.3 15.0 6.02414-13 14.8 5.8 32.7 34.9 11.8 6.0 2414-16 16.4 5.1 22.4 40.4 15.6 5.92414-2 17.0 4.7 23.8 38.4 16.1 5.9 2414-30 15.7 4.9 24.1 40.0 15.2 5.82414-20 15.9 5.4 23.9 39.1 15.7 5.8 2414-19 16.3 4.7 20.3 40.6 18.1 5.62414-1 16.1 4.8 21.8 40.3 17.1 5.6 2414-25 15.8 5.4 22.1 39.8 16.9 5.42414-24 15.6 6.4 24.8 35.7 17.6 5.3 2414-10 17.8 5.7 21.9 38.3 16.3 4.92414-17 16.6 4.9 22.6 38.8 17.1 4.9 2414-7 17.6 5.0 16.9 40.8 19.7 4.52414-18 16.7 5.7 18.7 39.9 19.0 4.4 2414-3 16.4 5.9 22.0 38.7 17.1 4.42414-5 16.4 3.9 14.4 42.6 22.7 4.3 2414-23 18.0 5.5 20.2 38.9 17.4 4.22414-22 16.3 5.4 21.4 38.1 18.8 4.0 2414-28 17.2 4.4 16.0 41.8 20.6 3.92414-21 17.6 5.0 18.0 40.3 19.0 3.7 2414-4 17.4 4.5 15.8 40.7 21.6 3.1Avg. 16.2 5.1 22.2 39.8 16.7 5.6

Codon optimization/motif-alteration of LS_DGAT2 resulted in a slightdecrease in activity and codon optimization/motif-alteration of PR_DGAT2did not substantially affect activity when expressed in soy.

Results for pKR1335 (MA_DGAT2; SEQ ID NO:256; Experiment MSE2427),pKR1419 (MA_DGAT2cod; SEQ ID NO:372; Experiment MSE2428), pKR1328(DH_DGAT2; SEQ ID NO:161; Experiment MSE2429) and pKR1420 (DH_DGAT2cod;SEQ ID NO:366; Experiment MSE2430) are shown in Tables 78, Table 79,Table 80 and Table 81, respectively.

TABLE 78 Oil concentrations and fatty acid profiles for events fromMSE2427 MSE2427 (MA DGAT2) Event 16:0 18:0 18:1 18:2 18:3 % Oil 2427-2413.8 6.4 30.3 38.6 10.8 9.6 2427-21 13.8 6.3 32.0 36.5 11.2 8.0 2427-514.8 5.9 25.2 41.7 12.3 7.7 2427-30 16.0 5.7 21.7 41.3 15.3 7.6 2427-315.8 6.5 24.1 40.0 13.6 7.2 2427-17 14.4 6.2 30.1 36.9 12.4 7.0 2427-2916.6 6.0 19.4 41.3 16.7 6.1 2427-27 16.6 5.8 18.2 42.5 16.9 6.1 2427-2515.5 7.0 24.2 38.3 15.0 5.9 2427-6 16.8 5.3 18.8 42.7 16.4 5.6 2427-816.1 6.3 19.8 40.8 17.0 5.4 2427-15 16.9 5.8 20.6 39.0 17.7 4.7 2427-1316.3 5.7 19.9 40.3 17.8 4.7 2427-9 15.5 5.2 21.7 38.5 19.1 4.6 2427-3115.7 6.2 17.9 41.2 19.1 4.5 2427-4 15.3 5.2 20.4 39.4 19.8 4.5 2427-1414.1 5.9 18.8 43.9 17.3 4.5 2427-19 15.6 6.2 22.7 37.6 17.9 4.5 2427-217.8 5.7 17.5 39.8 19.2 4.5 2427-7 17.0 5.3 17.6 41.6 18.5 4.4 2427-2016.9 5.5 18.8 39.2 19.7 4.3 2427-10 16.6 5.2 17.6 40.5 20.2 4.1 2427-2317.7 5.2 15.9 40.6 20.7 4.0 2427-11 15.5 5.3 23.4 39.9 15.9 4.0 2427-2816.7 5.3 16.2 40.1 21.7 3.8 2427-1 16.2 5.4 17.5 40.0 20.9 3.7 2427-1617.3 5.7 18.1 39.1 19.8 3.6 2427-26 16.8 5.1 16.7 40.9 20.5 3.4 2427-1216.9 4.7 13.5 40.4 24.3 3.2 Avg. 16.0 5.7 20.6 40.1 17.5 5.2

TABLE 79 Oil concentrations and fatty acid profiles for events fromMSE2428 MSE2428 (MA DGAT2cod) Event 16:0 18:0 18:1 18:2 18:3 % Oil2428-15 11.2 6.5 35.7 39.4 7.2 13.1 2428-25 12.5 5.7 31.0 40.8 10.1 11.82428-5 11.9 6.2 34.0 39.2 8.7 11.1 2428-20 12.4 6.4 33.4 38.9 8.9 11.12428-27 12.2 6.4 34.6 38.0 8.8 10.4 2428-1 11.4 7.1 36.9 36.2 8.4 10.32428-21 11.9 6.7 34.0 38.3 9.1 10.0 2428-17 13.4 6.0 28.8 41.5 10.4 10.02428-22 15.5 5.3 22.2 44.6 12.4 9.2 2428-3 12.8 6.5 31.8 37.4 11.6 8.52428-10 14.6 6.7 29.0 37.6 12.2 8.0 2428-12 13.1 6.6 30.8 39.6 9.8 7.32428-23 13.9 6.1 25.9 40.9 13.2 6.9 2428-29 12.4 7.3 35.0 35.7 9.6 6.72428-11 16.6 5.4 17.1 43.3 17.6 5.5 2428-30 16.8 8.7 23.0 38.3 13.1 5.52428-2 15.9 6.4 21.4 39.3 17.0 5.0 2428-13 17.2 5.5 16.2 42.4 18.6 4.82428-28 16.8 6.0 18.9 41.5 16.7 4.7 2428-31 17.0 5.7 16.4 41.9 19.0 4.32428-18 16.4 5.4 19.5 39.5 19.2 4.2 2428-6 16.4 4.8 16.4 41.4 20.9 3.92428-8 16.7 5.8 17.0 40.0 20.4 3.9 2428-4 16.6 5.4 16.9 41.9 19.2 3.82428-16 17.5 5.0 12.1 41.0 24.4 3.8 2428-9 17.1 5.2 14.8 40.9 21.9 3.82428-26 16.5 5.8 17.6 38.3 21.8 3.4 2428-19 16.8 4.9 13.4 39.8 25.1 3.32428-7 14.4 6.2 13.0 43.6 22.9 3.3 2428-24 17.8 5.4 18.7 39.5 18.5 2.8Avg. 14.9 6.0 23.9 40.0 15.2 6.7

TABLE 80 Oil concentrations and fatty acid profiles for events fromMSE2429 MSE2429 (DH DGAT2) Event 16:0 18:0 18:1 18:2 18:3 % Oil 2429-3016.6 5.2 17.9 44.0 16.3 6.7 2429-20 16.8 4.9 16.8 43.2 18.3 5.6 2429-1015.9 5.4 19.8 41.8 17.2 5.5 2429-29 17.0 5.7 18.9 41.1 17.3 5.5 2429-1316.6 5.1 18.0 42.7 17.7 5.3 2429-23 16.1 5.5 19.5 41.1 17.8 5.3 2429-2516.1 5.5 19.0 41.7 17.7 5.2 2429-26 16.1 5.6 18.9 40.9 18.5 5.1 2429-1416.0 5.3 19.5 41.1 18.1 4.9 2429-18 15.9 6.4 21.2 39.0 17.4 4.8 2429-2716.6 5.2 18.9 41.0 18.3 4.7 2429-17 16.9 4.9 17.1 42.0 19.1 4.7 2429-1115.2 4.5 17.3 43.6 19.3 4.7 2429-31 16.4 5.7 19.3 40.1 18.5 4.6 2429-716.3 5.7 19.6 39.6 18.9 4.2 2429-19 16.5 5.9 20.2 37.6 19.8 4.1 2429-116.3 4.8 15.5 41.3 22.1 4.0 2429-24 16.9 5.0 17.1 39.4 21.6 4.0 2429-516.7 5.5 19.5 39.8 18.4 3.9 2429-8 16.1 6.0 20.3 39.3 18.4 3.9 2429-317.2 5.5 18.1 40.2 19.0 3.8 2429-12 16.1 5.1 18.4 40.2 20.3 3.8 2429-2116.6 5.2 15.5 40.6 22.1 3.8 2429-6 16.3 5.1 17.6 40.4 20.5 3.6 2429-1517.2 4.5 12.7 42.6 23.0 3.3 2429-4 15.7 5.5 16.6 40.4 21.8 3.3 2429-2813.7 5.6 14.4 44.0 22.3 3.2 2429-16 16.1 4.6 15.8 39.8 23.6 3.1 2429-2214.6 4.8 11.2 43.6 25.7 3.0 2429-2 16.9 4.4 13.6 41.3 23.8 2.9 2429-916.9 5.0 16.6 38.6 22.9 2.7 Avg. 16.3 5.3 17.6 41.0 19.9 4.3

TABLE 81 Oil concentrations and fatty acid profiles for events fromMSE2430 MSE2430 (DH DGAT2cod) Event 16:0 18:0 18:1 18:2 18:3 % Oil2430-11 15.6 5.4 21.4 43.3 14.3 8.3 2430-7 15.7 5.6 21.4 44.0 13.2 8.02430-2 15.9 5.1 20.5 43.9 14.6 7.9 2430-14 16.5 4.7 18.4 46.2 14.3 7.82430-9 17.0 4.7 21.8 41.3 15.3 6.7 2430-8 16.1 4.9 19.1 45.2 14.7 6.72430-13 16.1 6.4 15.5 44.1 17.8 6.1 2430-6 16.0 6.2 26.8 37.8 13.3 6.02430-10 15.9 5.6 19.9 41.3 17.3 5.8 2430-4 16.4 5.6 18.7 41.9 17.3 5.32430-15 17.9 4.7 17.6 41.6 18.2 5.2 2430-20 16.0 5.5 20.4 39.6 18.5 5.12430-21 16.7 5.7 19.2 40.8 17.6 5.0 2430-3 16.7 5.5 18.3 42.6 16.9 4.92430-26 16.7 5.8 21.9 38.9 16.7 4.8 2430-27 17.0 5.1 17.6 40.7 19.7 4.72430-23 15.0 5.2 17.9 41.1 20.9 4.7 2430-22 15.8 5.9 21.8 38.9 17.6 4.62430-25 16.6 5.9 18.4 40.6 18.4 4.4 2430-16 16.7 4.9 15.8 42.5 20.0 4.32430-5 17.0 5.8 19.9 38.7 18.7 4.3 2430-18 16.8 5.4 18.4 40.2 19.1 4.02430-24 16.2 5.1 18.3 40.1 20.4 4.0 2430-1 17.0 5.0 16.9 40.2 21.0 3.62430-17 16.1 4.8 16.5 39.2 23.3 3.5 2430-19 17.9 4.7 13.6 39.0 24.9 3.32430-12 18.5 5.1 21.0 36.8 18.6 2.9 Avg. 16.5 5.3 19.1 41.1 17.9 5.3

Codon optimization/motif alteration of MA_DGAT2 and DH_DGAT2substantially increased activity when expressed in soy.

Results for pKR1332 (PA_DGAT2; SEQ ID NO:260; Experiment MSE2431),pKR1421 (PA_DGAT2cod; SEQ ID NO:365; Experiment MSE2432), pKR1324(TD_DGAT2A; SEQ ID NO:121; Experiment MSE2433) and pKR1422(TD_DGAT2Acod; SEQ ID NO:364; Experiment MSE2434) are shown in Tables82, Table 83, Table 84 and Table 85, respectively.

TABLE 82 Oil concentrations and fatty acid profiles for events fromMSE2431 MSE2431 (PA DGAT2) Event 16:0 18:0 18:1 18:2 18:3 % Oil 2431-2816.4 6.2 26.2 38.7 12.5 8.5 2431-21 16.2 4.4 20.4 45.0 14.0 8.0 2431-1915.3 4.9 20.3 43.7 15.8 7.9 2431-23 14.5 6.0 22.8 41.1 15.6 7.5 2431-2215.9 7.8 28.1 35.1 13.0 7.1 2431-6 15.4 6.0 24.2 38.7 15.6 6.8 2431-2516.8 5.0 22.7 40.5 15.0 6.5 2431-24 16.6 5.6 18.9 42.8 16.1 6.3 2431-716.3 4.4 15.8 45.1 18.3 6.1 2431-30 15.6 5.6 22.1 39.8 16.8 5.9 2431-3116.2 5.1 18.4 41.0 19.3 5.8 2431-11 16.2 5.8 18.9 42.2 16.9 5.7 2431-2615.7 5.5 22.2 39.5 17.1 5.7 2431-1 16.6 5.2 19.5 42.2 16.4 5.6 2431-215.9 5.1 18.8 41.1 19.2 5.5 2431-10 15.4 4.7 20.8 41.1 18.0 5.5 2431-515.8 4.7 19.4 41.1 19.1 5.5 2431-27 16.8 4.9 17.7 42.3 18.2 5.5 2431-917.0 5.7 16.7 38.8 21.8 5.0 2431-20 17.0 5.1 18.4 41.8 17.7 4.7 2431-1417.3 4.4 11.5 45.2 21.5 4.7 2431-17 18.0 4.3 13.2 42.6 22.0 4.5 2431-416.6 5.4 20.6 38.0 19.4 4.5 2431-15 15.2 5.3 21.3 39.7 18.6 4.4 2431-2917.2 5.1 18.2 41.1 18.3 4.3 2431-18 16.4 5.1 21.0 39.8 17.6 4.3 2431-1317.4 5.3 19.2 38.0 20.1 4.3 2431-12 18.3 4.4 15.5 40.3 21.6 4.2 2431-1618.0 5.1 16.5 38.9 21.6 4.2 2431-8 15.4 4.8 20.7 39.2 19.9 4.0 2431-315.9 5.3 21.7 39.9 17.2 2.9 Avg. 16.4 5.2 19.7 40.8 17.9 5.5

TABLE 83 Oil concentrations and fatty acid profiles for events fromMSE2432 MSE2432 (PA DGAT2cod) Event 16:0 18:0 18:1 18:2 18:3 % Oil2432-13 12.3 4.8 37.3 39.6 5.9 20.8 2432-6 13.2 4.3 29.8 44.0 8.7 17.42432-11 12.3 5.2 38.8 37.3 6.3 17.0 2432-16 12.4 5.8 42.9 33.1 5.8 16.02432-27 13.8 4.3 36.6 37.2 8.1 14.1 2432-21 14.1 4.7 26.6 44.3 10.2 13.92432-23 15.5 4.2 19.6 50.0 10.6 13.7 2432-5 15.2 4.8 27.3 42.1 10.5 13.42432-8 15.6 4.5 24.5 45.6 9.9 13.3 2432-15 12.2 6.2 44.0 31.1 6.5 13.22432-9 16.7 4.5 21.3 46.2 11.2 12.3 2432-4 15.8 4.4 22.0 46.5 11.4 12.12432-17 15.4 4.6 19.9 48.0 12.2 11.9 2432-12 15.2 4.8 22.7 45.7 11.611.3 2432-26 16.3 5.2 21.5 46.5 10.5 11.1 2432-25 14.0 6.5 30.0 39.9 9.711.0 2432-7 15.7 4.8 25.2 43.4 10.9 10.6 2432-3 14.8 5.1 24.7 44.2 11.210.4 2432-28 15.0 4.7 23.0 45.7 11.7 10.4 2432-29 15.5 5.4 21.9 45.012.3 9.7 2432-10 14.6 5.4 30.2 39.2 10.5 9.6 2432-18 16.2 5.1 21.0 44.013.7 8.4 2432-1 15.6 5.2 18.5 46.3 14.3 7.8 2432-30 15.2 4.5 22.7 43.613.9 7.6 2432-22 16.4 4.3 15.0 45.6 18.6 7.2 2432-31 15.9 4.4 17.9 45.416.4 7.1 2432-2 15.7 4.8 22.9 42.4 14.2 7.1 2432-20 16.0 4.5 28.5 36.614.5 6.9 2432-14 15.4 5.1 20.9 41.2 17.5 6.8 2432-19 17.4 4.9 14.7 42.720.3 5.3 2432-24 17.2 3.7 11.5 46.7 20.9 5.1 Avg. 15.1 4.9 25.3 42.911.9 11.1

TABLE 84 Oil concentrations and fatty acid profiles for events fromMSE2433 MSE2433 (TD DGAT2A) Event 16:0 18:0 18:1 18:2 18:3 % Oil 2433-4515.7 4.0 20.3 50.4 9.7 16.0 2433-16 15.5 4.5 21.3 49.5 9.3 15.0 2433-1914.5 4.7 22.8 48.3 9.7 14.9 2433-25 15.2 4.1 20.5 50.3 9.9 14.6 2433-2913.8 5.1 23.8 47.3 10.1 14.3 2433-21 15.0 4.8 22.1 48.0 10.1 14.02433-30 14.1 4.1 20.4 50.1 11.3 12.8 2433-38 16.0 3.5 16.9 52.2 11.412.8 2433-39 14.5 5.7 22.8 46.5 10.5 12.7 2433-42 14.6 5.4 23.2 46.210.5 11.8 2433-34 14.0 4.6 20.3 49.3 11.8 11.7 2433-26 13.8 5.1 21.648.1 11.4 11.6 2433-5 14.2 5.9 24.8 44.3 10.8 11.3 2433-11 15.5 5.0 21.646.8 11.2 10.9 2433-1 15.3 4.9 21.9 46.7 11.2 10.9 2433-15 14.0 6.3 26.642.3 10.8 10.6 2433-13 14.7 5.5 23.4 44.1 12.2 10.2 2433-3 15.2 5.2 23.445.1 11.1 10.1 2433-6 15.1 5.6 23.2 44.2 12.0 10.1 2433-24 16.1 4.3 19.047.1 13.5 9.6 2433-23 15.9 4.0 18.0 47.7 14.4 9.6 2433-43 14.9 5.5 22.544.7 12.5 9.5 2433-2 16.3 4.4 17.1 48.0 14.2 9.3 2433-4 14.2 6.1 23.444.0 12.4 9.0 2433-17 15.5 5.2 21.4 44.4 13.4 8.8 2433-7 15.8 4.8 18.746.7 14.0 8.8 2433-10 15.7 5.6 23.1 42.9 12.7 8.2 2433-14 16.4 4.8 18.946.0 13.9 8.1 2433-8 16.3 5.3 19.4 44.5 14.4 7.8 2433-12 16.0 6.3 22.342.6 12.8 7.1 2433-9 17.4 4.3 15.0 44.7 18.5 5.3 Avg. 15.2 5.0 21.3 46.512.0 10.9

TABLE 85 Oil concentrations and fatty acid profiles for events fromMSE2434 MSE2434 (TD DGAT2Acod) Event 16:0 18:0 18:1 18:2 18:3 % Oil2434-3 14.9 4.6 26.4 43.7 10.4 12.7 2434-12 15.0 5.1 22.3 45.5 12.1 11.12434-7 14.8 5.3 26.6 41.1 12.3 10.9 2434-20 13.9 6.2 23.8 43.9 12.3 10.32434-6 15.2 3.8 18.7 46.5 15.9 9.6 2434-16 14.7 5.0 22.6 44.0 13.8 9.62434-5 15.5 5.1 22.3 43.9 13.2 9.5 2434-24 14.8 6.4 25.9 40.9 12.1 9.42434-15 15.4 5.6 22.1 43.5 13.4 9.0 2434-9 13.4 4.3 18.3 47.0 17.0 8.82434-18 15.3 5.6 24.5 41.0 13.6 8.7 2434-13 15.7 5.0 18.5 46.9 13.8 8.62434-14 15.4 5.2 19.7 44.7 15.1 8.6 2434-10 14.7 6.8 24.3 39.5 14.8 8.52434-1 15.2 5.0 19.4 44.4 16.0 8.4 2434-28 14.5 6.1 24.8 40.9 13.6 8.22434-11 14.0 6.6 27.6 38.8 13.0 8.2 2434-8 15.4 5.7 22.0 42.4 14.4 8.02434-23 15.6 4.8 19.3 44.6 15.7 7.9 2434-27 15.4 5.2 23.4 40.9 15.1 7.82434-17 15.7 5.6 24.5 39.4 14.8 7.5 2434-25 15.5 5.0 20.6 41.4 17.4 7.02434-2 15.5 4.8 18.9 44.1 16.7 6.9 2434-4 16.7 4.3 18.3 46.3 14.5 6.82434-21 15.9 4.0 15.9 45.4 19.0 6.8 2434-31 15.5 4.5 19.4 42.1 18.6 6.72434-29 16.3 4.4 18.5 43.7 17.1 6.5 2434-26 16.1 5.1 18.7 40.1 20.0 5.92434-30 16.8 4.8 17.2 42.4 18.8 5.8 2434-22 16.7 4.1 15.1 46.0 18.1 5.72434-19 16.1 8.1 23.5 38.2 14.1 5.7 Avg. 15.3 5.2 21.4 43.0 15.1 8.2

Codon optimization/motif-alteration of PA_DGAT2 resulted in very largeincrease in activity and codon optimization/motif-alteration ofTD_DGAT2A decreased activity when expressed in soy.

Results for pKR1333 (RG_DGAT2; SEQ ID NO:266; Experiment MSE2447) andpKR1423 (RG_DGAT2cod; SEQ ID NO:371; Experiment MSE2448) are shown inTables 86 and Table 87, respectively.

TABLE 86 Oil concentrations and fatty acid profiles for events fromMSE2447 MSE2447 (RG DGAT2) Event 16:0 18:0 18:1 18:2 18:3 % Oil 2447-2716.8 6.3 31.2 34.2 11.5 6.4 2447-18 16.4 4.3 22.2 41.4 15.7 6.1 2447-1716.8 5.9 27.5 35.6 14.2 5.7 2447-28 17.1 4.8 24.0 38.2 15.9 5.3 2447-1017.5 5.9 25.8 37.2 13.7 5.2 2447-13 16.6 5.1 23.1 38.3 16.9 5.2 2447-717.2 4.9 21.1 39.4 17.3 4.9 2447-11 16.7 5.2 21.9 39.0 17.2 4.8 2447-2114.2 5.6 21.0 40.3 18.9 4.6 2447-24 17.6 5.2 22.9 37.7 16.6 4.6 2447-616.8 5.0 17.0 41.3 19.9 4.4 2447-14 17.2 4.4 16.3 40.1 22.0 4.4 2447-2517.2 5.2 19.6 39.7 18.4 4.4 2447-9 16.9 4.4 20.8 38.8 19.1 4.3 2447-816.7 5.4 24.4 36.1 17.4 4.2 2447-26 17.1 4.4 17.0 40.0 21.4 4.1 2447-2316.5 5.1 22.0 39.1 17.4 4.0 2447-16 16.4 4.8 20.4 38.1 20.2 3.9 2447-1916.7 4.6 19.3 39.2 20.3 3.9 2447-30 17.3 4.6 15.3 39.3 23.5 3.8 2447-416.3 5.0 22.2 36.6 19.8 3.7 2447-3 17.3 4.5 18.0 39.1 21.1 3.6 2447-2218.0 4.4 13.2 42.1 22.3 3.5 2447-15 19.5 5.2 21.8 32.8 20.6 3.3 2447-2017.9 5.0 15.6 40.0 21.6 3.1 2447-31 17.5 4.7 16.2 39.8 21.9 3.0 2447-1217.4 4.9 15.9 38.9 22.8 2.7 Avg. 17.0 5.0 20.6 38.6 18.8 4.3

TABLE 87 Oil concentrations and fatty acid profiles for events fromMSE2448 MSE2448 (RG DGAT2cod) Event 16:0 18:0 18:1 18:2 18:3 % Oil2448-6 16.0 6.1 31.7 33.5 12.7 6.7 2448-4 16.7 5.3 26.6 37.6 13.8 6.52448-5 16.1 5.2 28.2 36.7 13.8 6.0 2448-22 16.5 5.4 25.6 35.9 16.5 5.42448-17 15.8 5.9 29.4 35.1 13.9 5.2 2448-20 16.3 5.7 27.4 35.2 15.4 5.22448-16 17.4 4.6 17.9 42.8 17.3 5.0 2448-28 16.6 4.2 19.1 40.6 19.4 4.72448-13 17.2 4.9 24.1 37.1 16.7 4.7 2448-30 17.7 3.6 14.2 43.3 21.2 4.22448-2 17.3 5.2 21.9 38.0 17.6 3.9 2448-24 16.8 4.3 16.0 38.9 23.9 3.72448-11 17.8 4.7 14.2 40.8 22.5 3.6 2448-18 16.3 4.3 13.3 39.8 26.3 3.62448-29 17.1 4.9 19.1 37.8 21.1 3.5 2448-12 17.1 5.2 15.0 40.7 22.0 3.52448-3 16.3 4.5 14.8 39.1 25.3 3.5 2448-9 17.0 4.8 17.8 38.2 22.3 3.42448-25 17.9 4.1 17.1 40.2 20.6 3.3 2448-23 16.9 4.2 15.5 40.8 22.6 3.22448-14 16.8 6.0 17.4 38.3 21.5 3.0 2448-7 17.1 4.4 14.2 41.1 23.2 3.02448-19 16.7 4.0 13.5 42.5 23.3 2.9 2448-21 16.6 3.8 13.5 39.9 26.2 2.82448-1 16.8 4.5 16.5 38.4 23.8 2.2 2448-15 17.4 4.7 18.4 39.8 19.7 2.2Avg. 16.9 4.8 19.3 38.9 20.1 4.0

Codon optimization/motif-alteration of RG_DGAT2 did not substantiallyaffect activity when expressed in soy.

Results for pKR1375 (LS_DGAT1; SEQ ID NO:300; Experiment MSE2510),pKR1514 (LS_DGAT1cod; SEQ ID NO:374; Experiment MSE2511), pKR1334(MA_DGAT1; SEQ ID NO:280; Experiment MSE2512) and pKR1419 (MA_DGAT2cod;SEQ ID NO:372; Experiment MSE2513) are shown in Tables 88, Table 89,Table 90 and Table 91, respectively.

TABLE 88 Oil concentrations and fatty acid profiles for events fromMSE2510 MSE2510 (LS DGAT1) Event 16:0 18:0 18:1 18:2 18:3 % Oil 2510-1415.1 6.4 30.1 38.4 10.0 9.8 2510-27 15.3 6.1 27.3 39.8 11.6 9.0 2510-1315.6 6.8 27.7 37.7 12.1 8.4 2510-25 18.5 6.6 24.7 37.0 13.1 8.0 2510-1115.9 6.5 27.2 36.8 13.6 7.1 2510-4 16.3 5.3 25.3 37.5 15.6 7.1 2510-2316.2 5.9 23.9 38.9 15.1 6.4 2510-2 16.5 5.9 21.2 39.8 16.5 6.3 2510-1517.0 5.5 19.0 43.3 15.2 6.2 2510-6 18.7 5.6 22.7 39.1 13.9 6.0 2510-1915.5 6.0 22.0 40.2 16.3 5.9 2510-22 16.6 6.8 19.4 41.5 15.6 5.9 2510-916.5 5.6 21.3 39.8 16.8 5.9 2510-28 16.7 5.6 20.0 40.6 17.0 5.7 2510-2017.0 5.8 20.9 41.0 15.3 5.6 2510-8 16.9 5.9 20.3 40.6 16.3 5.2 2510-1217.1 5.7 20.7 38.9 17.6 5.2 2510-24 16.9 6.5 24.0 37.8 14.7 5.2 2510-1617.0 6.2 22.5 37.4 16.9 4.9 2510-31 16.9 5.9 19.9 40.1 17.3 4.8 2510-1717.4 5.3 20.8 38.6 17.9 4.5 2510-21 16.8 5.1 18.0 40.9 19.2 4.4 2510-1817.5 5.1 17.9 39.8 19.7 4.3 2510-10 16.2 4.8 17.0 42.8 19.2 4.2 2510-3017.3 4.9 16.5 41.3 19.9 4.2 2510-26 17.7 5.2 20.4 38.2 18.5 4.1 2510-517.8 4.4 13.9 41.7 22.1 3.9 2510-1 16.8 4.8 17.2 40.1 21.2 3.9 2510-717.4 5.6 18.2 39.6 19.3 3.5 2510-3 18.2 4.5 14.1 40.7 22.5 3.1 2510-2917.1 4.3 13.2 40.3 25.1 2.5 Avg 16.8 5.6 20.9 39.7 16.9 5.5

TABLE 89 Oil concentrations and fatty acid profiles for events fromMSE2511 MSE2511 (LS DGAT1cod) Event 16:0 18:0 18:1 18:2 18:3 % Oil2511-19 14.6 6.8 31.3 38.6 8.7 12.2 2511-18 16.0 8.2 30.7 36.7 8.4 11.92511-29 15.0 6.4 29.1 37.9 11.6 9.2 2511-6 15.9 5.7 24.8 40.8 12.6 8.02511-8 17.6 6.5 25.3 38.6 12.0 7.9 2511-21 17.1 4.7 16.6 45.9 15.6 7.02511-2 15.9 6.5 24.5 39.0 14.1 6.9 2511-27 16.9 5.9 22.1 39.9 15.2 6.52511-12 15.8 6.5 22.9 39.0 15.7 6.4 2511-23 15.7 6.8 29.6 36.6 11.3 6.42511-20 16.8 6.0 20.5 40.4 16.3 6.3 2511-5 15.8 5.8 24.7 37.7 16.0 6.12511-30 16.5 6.7 23.8 39.3 13.6 5.6 2511-13 17.3 5.4 20.1 39.4 17.8 5.42511-4 16.9 6.1 21.7 39.3 15.9 5.2 2511-15 16.9 5.4 20.6 40.2 17.0 5.02511-10 16.9 5.5 20.9 39.1 17.7 4.9 2511-25 17.2 5.9 20.7 38.1 18.1 4.82511-3 16.7 5.7 19.6 40.5 17.5 4.8 2511-17 17.3 5.2 19.1 40.0 18.3 4.72511-7 17.3 5.4 19.7 40.7 16.8 4.5 2511-14 17.2 5.9 19.2 40.4 17.3 4.32511-26 17.6 5.3 17.2 39.6 20.3 4.2 2511-9 17.5 5.3 17.8 40.3 19.0 4.12511-22 17.2 6.7 23.6 36.8 15.7 3.7 2511-16 17.3 5.5 16.0 41.1 20.0 3.72511-24 17.3 5.3 17.2 40.3 20.0 3.7 2511-28 17.4 4.9 14.3 43.5 20.0 3.72511-1 17.1 5.4 18.6 40.2 18.7 3.6 2511-31 15.9 4.8 15.4 39.8 24.1 2.92511-11 16.7 4.2 14.1 41.7 23.4 2.9 Avg. 16.7 5.8 21.3 39.7 16.4 5.7

TABLE 90 Oil concentrations and fatty acid profiles for events fromMSE2512 MSE2512 (MA DGAT1) Event 16:0 18:0 18:1 18:2 18:3 % Oil 2512-2711.1 6.2 42.7 34.0 6.1 13.6 2512-5 12.0 6.6 35.4 37.9 8.1 13.5 2512-212.9 8.2 35.3 36.5 7.1 12.4 2512-24 12.7 6.6 35.5 37.2 8.0 11.6 2512-1413.5 6.2 32.4 38.4 9.6 11.3 2512-1 14.6 6.5 30.8 38.4 9.6 11.0 2512-413.0 7.2 37.2 34.5 8.1 11.0 2512-15 13.5 5.7 32.5 37.7 10.6 10.2 2512-812.6 6.2 36.7 36.0 8.5 10.1 2512-25 13.8 5.9 31.1 37.5 11.7 8.9 2512-316.1 5.7 24.7 39.2 14.3 6.6 2512-9 16.2 5.9 20.3 42.0 15.6 6.5 2512-1916.4 5.9 23.1 39.5 15.2 6.1 2512-7 16.5 6.4 23.1 38.7 15.3 6.0 2512-1116.1 5.5 21.5 40.0 16.9 5.9 2512-18 16.3 5.9 20.1 41.3 16.4 5.6 2512-3017.4 4.8 16.7 42.7 18.4 5.4 2512-10 17.0 5.2 18.1 42.0 17.6 5.4 2512-2117.0 5.2 18.1 41.9 17.9 5.3 2512-13 17.0 4.5 18.2 40.6 19.7 5.1 2512-3116.7 5.2 19.4 41.3 17.4 4.9 2512-12 17.8 4.6 17.7 43.2 16.6 4.8 2512-2617.7 4.8 16.9 42.4 18.3 4.8 2512-22 17.3 4.4 15.2 40.9 22.1 4.6 2512-1716.9 5.4 18.1 39.5 20.1 4.5 2512-20 17.2 5.5 20.1 38.5 18.8 4.0 2512-1616.4 4.5 17.5 40.3 21.2 4.0 2512-29 16.8 4.4 15.5 40.4 22.8 3.8 2512-2317.9 5.1 15.5 40.6 20.9 3.1 2512-28 17.3 6.1 20.4 38.8 17.3 2.6 Avg.15.6 5.7 24.3 39.4 15.0 7.1

TABLE 91 Oil concentrations and fatty acid profiles for events fromMSE2513 MSE22513 (MA DGAT1cod) Event 16:0 18:0 18:1 18:2 18:3 % Oil2513-18 11.9 6.2 36.9 38.8 6.2 17.6 2513-14 10.5 6.9 40.7 35.9 6.0 16.72513-12 11.2 7.3 41.4 34.3 5.8 15.0 2513-22 11.0 5.0 38.8 37.7 7.5 14.72513-31 12.2 6.4 36.2 37.8 7.4 13.5 2513-20 11.7 6.4 36.4 38.3 7.3 13.12513-19 12.6 5.1 33.6 39.4 9.2 13.0 2513-5 12.7 6.6 34.9 37.4 8.3 12.22513-13 14.7 6.2 30.6 37.8 10.6 10.0 2513-25 15.3 6.9 28.6 37.3 12.0 9.42513-30 14.8 7.6 29.6 36.1 11.9 8.7 2513-15 13.0 5.6 35.3 36.0 10.1 8.42513-11 15.2 7.3 28.5 37.6 11.4 8.2 2513-29 15.3 5.7 22.6 42.4 13.9 7.42513-2 16.6 6.1 21.7 41.7 13.9 7.1 2513-7 16.1 7.4 28.0 35.9 12.6 7.02513-24 15.4 5.8 25.0 39.3 14.5 6.7 2513-8 16.9 4.6 21.3 42.5 14.7 6.62513-23 15.7 6.7 27.7 36.3 13.6 6.5 2513-6 16.6 6.4 23.5 38.6 14.9 6.32513-3 16.6 7.6 25.6 37.2 13.1 6.1 2513-27 16.7 6.1 21.2 40.5 15.4 5.82513-16 15.5 5.9 19.2 43.5 15.9 5.7 2513-17 17.0 5.7 20.6 39.6 17.2 5.52513-28 17.3 5.5 20.4 40.2 16.6 5.4 2513-21 15.7 6.9 23.6 35.4 18.3 5.12513-4 16.4 5.0 20.3 41.8 16.5 5.0 2513-26 17.7 5.6 19.9 41.1 15.7 4.92513-9 17.6 4.9 16.1 44.2 17.2 4.9 2513-1 19.0 6.0 19.8 38.4 16.9 4.62513-10 16.4 5.2 21.8 38.1 18.6 4.0 Avg. 15.0 6.1 27.4 38.7 12.7 8.5

Codon optimization of LS_DGAT1 and MA_DGAT1 substantially increasedactivity when expressed in soy.

Results for pKR1392 (CZ_DGAT2; SEQ ID NO:355; Experiment MSE2520),pKR1512 (CZ_DGAT2cod; SEQ ID NO:367; Experiment MSE2521), pKR1409(MC_DGAT2; SEQ ID NO:360; Experiment MSE2522) and pKR1513 (MC_DGAT2cod;SEQ ID NO:369; Experiment MSE2523) are shown in Tables 92, Table 93,Table 94 and Table 95, respectively.

TABLE 92 Oil concentrations and fatty acid profiles for events fromMSE2520 MSE2520 (CZ DGAT2) Event 16:0 18:0 18:1 18:2 18:3 % Oil 2520-1915.4 5.6 20.5 44.7 13.8 8.7 2520-4 16.4 5.5 20.7 40.0 17.4 5.5 2520-1816.4 5.2 21.3 38.5 18.7 4.5 2520-23 17.2 5.7 18.8 39.8 18.5 4.4 2520-2816.9 6.6 22.3 37.6 16.6 4.4 2520-10 17.3 5.2 20.1 39.1 18.2 4.2 2520-817.4 6.1 21.8 37.5 17.2 4.2 2520-13 18.0 6.0 20.0 38.4 17.5 4.2 2520-1717.8 6.6 21.9 37.4 16.3 4.1 2520-14 17.2 6.6 24.9 34.4 16.9 4.1 2520-2217.1 6.5 21.3 37.5 17.6 4.0 2520-25 16.2 5.3 21.9 37.2 19.4 4.0 2520-2717.0 7.3 22.3 36.7 16.7 4.0 2520-3 16.9 4.3 16.3 41.0 21.6 3.9 2520-2916.6 5.6 19.6 38.3 20.0 3.8 2520-9 17.0 5.4 20.7 38.0 18.9 3.8 2520-1616.5 4.8 18.9 39.7 20.1 3.8 2520-24 16.7 5.8 21.3 37.8 18.4 3.7 2520-1217.0 5.3 17.7 40.1 19.9 3.6 2520-7 18.2 5.2 15.8 41.3 19.4 3.6 2520-516.5 5.8 20.9 37.6 19.2 3.5 2520-15 17.2 4.7 15.0 39.8 23.3 3.3 2520-617.4 6.6 20.0 38.7 17.4 3.3 2520-21 17.2 5.3 17.4 39.3 20.9 3.3 2520-2616.1 5.2 18.0 38.5 22.2 3.3 2520-20 16.7 5.9 17.4 38.9 21.1 3.3 2520-1116.4 4.9 16.4 39.2 23.0 3.0 2520-30 17.6 5.9 20.1 38.0 18.4 3.0 2520-216.9 5.2 17.2 39.2 21.5 2.8 2520-1 20.9 4.5 15.1 38.2 21.3 2.8 Avg. 17.15.6 19.5 38.7 19.0 3.9

TABLE 93 Oil concentrations and fatty acid profiles for events fromMSE2521 MSE2521 (CZ DGAT2cod) Event 16:0 18:0 18:1 18:2 18:3 % Oil2521-9 16.5 6.5 21.2 41.2 14.6 6.9 2521-19 16.2 6.8 23.0 38.5 15.5 5.82521-23 17.3 6.1 23.9 37.5 15.2 5.7 2521-30 17.0 6.0 21.2 38.3 17.5 5.62521-14 16.3 6.7 24.7 37.3 14.9 5.5 2521-20 16.6 6.1 24.0 38.1 15.2 5.52521-13 15.9 6.0 22.5 39.4 16.1 5.5 2521-10 16.4 5.7 23.7 39.0 15.3 5.52521-29 17.1 5.9 21.1 39.3 16.6 5.2 2521-3 16.7 5.5 19.9 40.2 17.7 5.02521-26 16.4 6.7 22.4 38.5 16.0 4.9 2521-31 17.2 6.1 19.9 38.9 17.9 4.82521-24 16.7 5.8 21.0 38.8 17.7 4.8 2521-6 17.1 5.4 21.4 37.8 18.3 4.72521-12 16.4 5.6 23.0 37.7 17.3 4.7 2521-5 16.7 6.4 23.3 37.2 16.4 4.52521-11 16.9 5.6 20.0 38.5 19.1 4.4 2521-2 17.1 5.7 20.2 39.8 17.3 4.42521-8 18.1 6.0 20.3 38.0 17.6 4.2 2521-16 17.7 5.2 19.1 38.9 19.0 4.12521-1 17.8 6.4 22.1 36.0 17.7 4.0 2521-21 17.1 4.8 17.9 39.7 20.4 3.82521-17 17.7 5.0 19.2 38.8 19.4 3.8 2521-28 16.0 4.6 18.9 40.3 20.2 3.52521-27 17.0 5.0 17.7 38.7 21.6 3.5 2521-22 17.5 4.6 17.1 39.7 21.2 3.32521-25 17.3 4.9 17.4 38.7 21.7 3.0 2521-7 17.6 4.7 14.7 39.7 23.3 3.02521-18 17.4 4.8 18.4 38.1 21.3 2.9 2521-4 17.1 4.9 16.4 38.6 23.1 2.82521-15 20.5 4.5 12.8 37.4 24.8 2.4 Avg. 17.1 5.6 20.3 38.7 18.4 4.4

TABLE 94 Oil concentrations and fatty acid profiles for events fromMSE2522 MSE2522 (MC DGAT2) Event 16:0 18:0 18:1 18:2 18:3 % Oil 2522-215.1 5.1 20.2 44.4 15.1 8.6 2522-28 14.9 6.7 25.1 38.1 15.1 6.3 2522-514.4 5.1 19.1 41.7 19.7 5.7 2522-19 15.8 7.7 28.2 35.0 13.3 5.6 2522-2116.4 7.2 21.9 37.8 16.7 5.2 2522-23 17.0 4.8 19.7 41.0 17.4 5.2 2522-615.9 5.8 20.0 40.4 17.8 5.2 2522-1 17.3 6.4 19.3 36.6 20.5 4.8 2522-1715.0 5.4 19.0 42.1 18.6 4.7 2522-18 16.0 5.3 18.0 39.6 21.0 4.7 2522-1115.0 5.3 19.0 40.4 20.3 4.6 2522-12 15.5 4.9 16.1 40.8 22.8 4.5 2522-816.7 8.5 21.6 37.8 15.5 4.4 2522-16 14.8 6.5 21.6 38.2 18.9 4.4 2522-2216.6 5.9 20.4 38.4 18.8 4.2 2522-9 15.9 7.1 19.2 40.0 17.8 4.2 2522-2914.6 4.7 16.8 41.0 22.8 4.0 2522-26 16.9 4.2 14.7 42.2 22.1 4.0 2522-1413.4 3.9 13.6 40.6 28.5 3.9 2522-30 16.0 4.8 20.3 36.7 22.2 3.9 2522-1515.3 5.4 16.7 39.7 22.9 3.7 2522-10 14.1 4.1 13.9 40.6 27.3 3.7 2522-415.9 4.4 14.6 43.3 21.8 3.5 2522-25 15.7 6.1 22.0 36.2 20.1 3.5 2522-2716.2 5.2 14.4 42.1 22.1 3.5 2522-3 16.4 7.2 20.7 39.9 15.8 3.5 2522-3115.9 5.7 19.8 39.1 19.5 3.4 2522-13 15.2 5.5 15.1 39.5 24.7 3.2 2522-2417.7 6.2 21.2 38.1 16.7 3.2 2522-20 15.4 5.3 15.5 39.6 24.3 2.9 2522-717.2 3.6 9.8 41.9 27.5 2.8 Avg. 15.7 5.6 18.6 39.8 20.2 4.4

TABLE 95 Oil concentrations and fatty acid profiles for events fromMSE2523 MSE2523 (MC DGAT2cod) Event 16:0 18:0 18:1 18:2 18:3 % Oil2523-1 14.7 5.8 24.2 42.7 12.7 8.0 2523-5 15.4 5.7 27.2 39.9 11.9 8.02523-25 16.0 5.5 23.7 40.3 14.5 6.5 2523-18 16.4 5.9 23.1 39.3 15.3 6.02523-20 15.8 5.8 24.7 39.0 14.7 6.0 2523-19 17.2 5.8 23.7 37.6 15.7 5.52523-2 16.8 6.2 22.1 38.7 16.2 5.3 2523-23 16.4 5.4 24.3 38.6 15.3 5.22523-16 17.0 5.7 22.5 38.5 16.4 5.1 2523-17 16.8 5.5 23.4 37.7 16.7 5.12523-7 17.4 5.4 17.6 43.7 15.9 5.1 2523-22 17.0 5.6 21.7 38.8 17.0 4.72523-4 16.5 7.0 19.6 40.1 16.8 4.6 2523-29 17.3 5.4 20.5 39.0 17.7 4.52523-6 17.2 5.9 23.3 37.6 16.0 4.5 2523-27 17.2 5.2 20.1 41.3 16.2 4.52523-28 16.8 5.3 21.4 38.8 17.7 4.5 2523-13 17.1 5.4 19.6 39.6 18.3 4.42523-14 17.2 5.2 21.0 37.9 18.7 4.1 2523-21 16.4 5.4 18.9 39.7 19.6 3.92523-30 16.8 5.6 16.7 40.0 21.0 3.8 2523-15 17.6 4.8 17.1 40.2 20.3 3.82523-8 17.2 5.5 17.5 41.6 18.2 3.7 2523-10 17.8 5.0 18.5 38.8 19.9 3.52523-3 17.3 4.7 17.2 39.2 21.5 3.5 2523-12 17.8 4.6 15.2 38.5 23.8 3.02523-9 16.6 4.3 15.0 39.8 24.2 2.9 2523-26 17.4 5.8 19.6 38.7 18.5 2.5Avg. 16.8 5.5 20.7 39.5 17.5 4.7

Codon optimization of CZ_DGAT2 and MC_DGAT2 slightly increased activitywhen expressed in soy.

Results for pKR1427 (CC_DGAT2; SEQ ID NO:361; Experiment MSE2525) andpKR1522 (CC_DGAT2cod; SEQ ID NO:373; Experiment MSE2526) are shown inTables 96 and Table 97, respectively.

TABLE 96 Oil concentrations and fatty acid profiles for events fromMSE2525 MSE2525 (CC DGAT2) Event 16:0 18:0 18:1 18:2 18:3 % Oil 2525-2016.1 5.6 19.2 44.2 14.9 7.7 2525-2 16.6 6.1 20.6 42.2 14.4 6.2 2525-816.5 5.6 21.7 41.3 15.0 5.9 2525-10 16.6 6.9 26.5 35.8 14.3 5.9 2525-1317.2 5.0 17.7 42.3 17.8 5.2 2525-21 17.2 6.7 24.2 35.9 16.1 5.1 2525-2616.7 6.6 23.8 38.1 14.8 5.0 2525-14 16.6 6.1 21.3 38.8 17.2 4.9 2525-2717.3 6.4 23.0 37.6 15.7 4.7 2525-30 18.0 4.8 15.2 42.5 19.4 4.7 2525-2516.7 4.9 18.7 39.7 20.1 4.5 2525-7 17.6 5.7 22.5 37.5 16.8 4.4 2525-417.4 5.5 19.2 41.0 16.9 4.4 2525-22 19.4 4.9 18.4 38.0 19.3 4.4 2525-1817.5 5.9 21.2 37.5 17.9 4.4 2525-3 17.6 4.8 17.5 37.7 22.3 4.1 2525-1115.8 5.1 20.6 42.0 16.6 4.0 2525-29 18.4 4.9 16.0 42.6 18.1 3.9 2525-917.5 5.3 18.6 40.0 18.6 3.9 2525-19 16.7 5.6 20.4 38.3 19.0 3.9 2525-617.5 5.4 19.7 38.0 19.4 3.8 2525-24 17.3 5.3 18.7 39.7 19.1 3.7 2525-1617.4 4.6 15.7 41.3 21.1 3.7 2525-15 16.8 4.5 16.9 39.4 22.4 3.6 2525-117.6 5.5 18.8 40.9 17.1 3.6 2525-5 16.2 4.9 17.1 40.5 21.2 3.4 2525-1718.7 5.1 15.9 38.0 22.4 2.5 Avg. 17.2 5.5 19.6 39.7 18.1 4.5

TABLE 97 Oil concentrations and fatty acid profiles for events fromMSE2526 MSE2526 (CC DGAT2cod) Event 16:0 18:0 18:1 18:2 18:3 % Oil2526-29 15.7 6.6 26.0 38.3 13.4 7.5 2526-28 15.3 5.8 28.9 36.9 13.2 7.02526-10 17.1 6.1 21.5 40.5 14.7 6.7 2526-5 15.5 5.8 27.8 36.6 14.3 6.62526-14 16.4 6.7 25.1 36.9 14.9 6.1 2526-22 17.1 5.7 20.9 40.7 15.5 6.12526-2 16.8 7.4 23.1 38.0 14.7 6.0 2526-6 16.7 6.6 25.0 38.0 13.7 5.82526-17 17.2 5.4 20.3 38.9 18.2 5.4 2526-7 17.2 5.4 19.8 40.5 17.0 5.32526-3 17.9 6.4 23.2 36.2 16.3 5.3 2526-23 17.0 7.0 24.2 36.4 15.4 5.22526-20 16.1 5.1 23.4 38.1 17.3 5.2 2526-9 17.1 6.7 24.8 35.6 15.9 5.22526-27 17.6 5.7 20.5 38.3 17.9 4.9 2526-26 17.1 6.9 24.7 37.4 13.9 4.82526-8 16.8 5.6 24.1 37.3 16.2 4.8 2526-19 15.6 6.1 25.4 36.1 16.8 4.82526-12 19.9 6.1 21.5 34.1 18.4 4.7 2526-21 18.2 5.9 18.6 39.2 18.2 4.62526-16 17.8 5.0 16.3 40.4 20.5 4.3 2526-11 17.1 6.8 23.2 36.3 16.7 4.32526-1 17.4 5.9 20.2 38.5 18.0 4.0 2526-4 17.1 7.0 25.6 34.9 15.3 3.72526-24 17.8 4.7 13.0 44.3 20.1 3.3 2526-15 17.9 5.0 16.8 39.3 20.9 3.32526-18 17.7 5.2 16.0 39.2 21.9 3.2 Avg. 17.1 6.0 22.2 38.0 16.6 5.1

Codon optimization of CC_DGAT2 slightly increased activity whenexpressed in soy.

A summary for expression of all wild-type and codon-optimized and/ormotif-altered DGATs in soy somatic embryos relative to wild-type YLDGAT2 is shown in FIG. 10. A codon optimized/motif-altered version ofTD_DGAT2b was not tested as signified by ND in FIG. 10. For FIG. 10,each wild-type gene was tested as a set of experiments along withYL_DGAT2 as control and a percent activity relative to YL_DGAT2 wasdetermined. For this, the top 5 events having highest oil concentrationsfrom each experiment were first averaged then normalized relative to theaverage of the top 5 events for wild-type YL_DGAT2 in that experiment(Avg. % Oil of top 5 events for a given DGAT/Avg. % Oil of top 5 eventsfor YL_DGAT2 (in that experimental set)×100%). For codon optimized/motifaltered genes, a relative activity factor was determined forcodon-optimized relative to wild-type activity in that experiment. Forthis, the top 5 events having highest oil concentrations were firstaveraged and then normalized relative to the average of the top 5 eventsfor the wild-type gene in that experimental set (Avg. % Oil of top 5events for codon-optimized and/or motif-altered DGAT/Avg. % Oil of top 5events for wild-type DGAT). The codon-optimized/motif-altered relativeactivity factor was then multiplied by the specific gene % activityrelative to YL_DGAT2 to normalize the codon-optimized/motif-alteredactivity to YL_DGAT2 (% activity relative to YL_DGAT2 x codon-optimizedrelative activity factor). In FIG. 10 for each DGAT gene, % activityrelative to YL_DGAT2 (solid black bar) for wild-type is displayed first(checkered bars) followed by relative % activity for codon-optimizedand/or motif-altered (white, or speckled bars). Relative activities forDGAT1 constructs are shown in the last three sets of FIG. 10.

MEGA

What is claimed is:
 1. An isolated polynucleotide comprising: (a) anucleotide sequence encoding a polypeptide having diacylglycerolacyltransferase activity wherein the polypeptide has at least 80% aminoacid identity, based on the Clustal V method of alignment, when comparedto an amino acid sequence as set forth in SEQ ID NOs:135, 136, 162, 176,215, 234, 265, 272, 279, 299, 304, 306, 310, 312, 314, 316, 318, 320,322, 351, or 363; (b) a nucleotide sequence encoding a polypeptidehaving diacylglycerol acyltransferase activity, wherein the nucleotidesequence has at least 80% sequence identity, based on the BLASTN methodof alignment, when compared to a nucleotide sequence as set forth in SEQID NO: 133, 134, 161, 175, 214, 233, 264, 271, 278, 298, 301, 303, 305,309, 311, 313, 315, 317, 319, 321, 350, or 352; (c) a nucleotidesequence encoding a polypeptide having diacylglycerol acyltransferaseactivity, wherein the nucleotide sequence hybridizes under stringentconditions to a nucleotide sequence as set forth in SEQ ID NO: 133, 134,161, 175, 214, 233, 264, 271, 278, 298, 301, 303, 305, 309, 311, 313,315, 317, 319, 321, 350, or 352; or (d) a complement of the nucleotidesequence of (a), (b) or (c), wherein the complement and the nucleotidesequence consist of the same number of nucleotides and are 100%complementary.
 2. The polypeptide of claim 1, wherein the amino acidsequence of the polypeptide has at least 85% sequence identity, based onthe Clustal V method of alignment, when compared to SEQ ID NO: 135, 136,162, 176, 215, 234, 265, 272, 279, 299, 304, 306, 310, 312, 314, 316,318, 320, 322, 351, or
 363. 3. The polypeptide of claim 1, wherein theamino acid sequence of the polypeptide has at least 90% sequenceidentity, based on the Clustal V method of alignment, when compared toSEQ ID NO: 135, 136, 162, 176, 215, 234, 265, 272, 279, 299, 304, 306,310, 312, 314, 316, 318, 320, 322, 351, or
 363. 4. The polypeptide ofclaim 1, wherein the amino acid sequence of the polypeptide has at least95% sequence identity, based on the Clustal V method of alignment, whencompared to SEQ ID NO: 135, 136, 162, 176, 215, 234, 255, 265, 272, 279,299, 304, 306, 310, 312, 314, 316, 318, 320, 322, 351, or
 363. 5. Theisolated polynucleotide of claim 1, wherein the nucleotide sequencecomprises SEQ ID NO: 133, 134, 161, 175, 214, 233, 264, 271, 278, 298,301, 303, 305, 309, 311, 313, 315, 317, 319, 321, 350, or
 352. 6. Theisolated polynucleotide of claim 1, wherein said polynucleotide isobtained from one or more oleaginous organisms.
 7. The isolatedpolynucleotide of claim of claim 6, wherein the oleaginous organism isselected from the group consisting of Torulaspora delbrueckii, Pichiaanomala, Debaryomyces hansenii, Candida zeylanoides, Lipomyces starkeyi,Mucor circinelloides, Phaffia rhodozyma, Rhodotorula glutinis,Mortierella alpina, and Cryptococcus curvatus.
 8. A recombinant DNAconstruct comprising the isolated nucleic acid fragment of claim 1operably linked to at least one regulatory sequence.
 9. A cellcomprising in its genome the recombinant DNA construct of claim
 8. 10.The cell of claim 9, wherein said cell is an oilseed plant cell.
 11. Atransgenic oilseed comprising the recombinant DNA construct of claim 8in its genome.
 12. The transgenic oilseed of claim 11, wherein saidoilseed is selected from the group consisting of soybean, corn, canola,sunflower, flax, cotton, and safflower.
 13. A method for increasing thetotal fatty acid content of an oilseed comprising: (a) transforming atleast one oilseed cell with the recombinant construct of claim 8; and,(b) selecting the transformed oilseed cell(s) of step (a) having anincreased total fatty acid content when compared to the total fatty acidcontent of a non-transgenic, null segregant oilseed.
 14. The method ofclaim 13, wherein the oilseed is selected from the group consisting ofsoybean, corn, canola, sunflower, flax, cotton, and safflower.
 15. Aproduct or by-product obtained from the transgenic oilseed of claim 11.16. A progeny plant obtained from the transgenic oilseed of claim 11.17. The cell of claim 9, wherein said cell is a fungal cell oroleaginous microbial organism.
 18. The fungal cell of claim 17, whereinsaid fungal cell is selected from the group consisting of Yarrowia,Candida, Rhodotorula, Rhodosporidium, Cryptococcus, Trichosporon andLipomyces.